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Tolerance of clinical vancomycin-resistant Enterococcus faecium isolates against UV-C light from a mobile source

Tolerance of clinical vancomycin-resistant Enterococcus faecium isolates against UV-C light from... Background Admission to a room previously occupied by patients carrying environmentally robust pathogens implies an increased risk of acquiring those pathogens. Therefore, ‘No-touch’ automated room disinfection systems, including devices based on UV-C irradiation, are discussed to improve terminal cleaning. It is still unclear if clinical isolates of relevant pathogens behave differently under UV-C irradiation compared to laboratory strains used in the approval process of disinfection procedures. In this study we analysed the susceptibility of well characterized clonally divergent vancomycin-resistant enterococci ( VRE) strains, including a linezolid-resistant isolate, against UV-C radiation. Methods Susceptibility against UV-C of ten clonally divergent clinical isolates of VRE was determined in comparison to the commonly used test organism Enterococcus hirae ATCC 10541. Ceramic tiles contaminated with 10 to 10 colony forming units/25 cm² of the different enterococci were positioned at a distance of 1.0 and 1.5 m and irradiated for 20 s, resulting in a UV-C dose of 50 and 22 mJ/cm², respectively. Reduction factors were calculated after quantitative culture of the bacteria recovered from treated and untreated surfaces. Results Susceptibility to UV-C varied considerably among the strains studied, with the mean value of the most robust strain being up to a power of ten lower compared to the most sensitive strain at both UV-C doses. The two most tolerant strains belonged to MLST sequence types ST80 and ST1283. The susceptibility of the laboratory strain E. hirae ATCC 10541 ranged between the most sensitive and most tolerant isolates for both irradiation doses. However, for UV-C dose of 22 mJ/cm², the reduction of the most tolerant isolate of ST1283 was statistically significantly lower compared to E. hirae ATCC 10541. The most susceptible strains belonged to the MLST sequence types ST117 and ST203. Conclusions These results indicate that UV-C doses reported in the literature are sufficient for the reduction of commonly used reference strains of enterococci but could be insufficient for the reduction of tolerant patient VRE- isolates in a hospital setting. Therefore, for future studies, the most tolerant clinical isolates should be used to validate automated UV-C devices or longer exposure times should be expected to ensure efficacy in the real world. Keywords Automatic room decontamination, Disinfection, UV-C, UV-irradiation, vancomycin resistant enterococci *Correspondence: Institute for Medical Microbiology, Virology and Hygiene, Department J. K. Knobloch Infection Prevention and Control, University Medical Center Hamburg- j.knobloch@uke.de Eppendorf, Hamburg, Germany Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 2 of 7 Background clinical environment to confirm efficacy [ 18]. For these In recent decades, contamination of surfaces in the efficacy tests, it is important to consider that recent clini - patient environment as cause for acquisition of noso- cal isolates may behave differently compared to labo - comial infections and transmission of pathogens has ratory strains, which have been commonly used in the gained importance. Especially for Staphylococcus aureus, approval process of disinfection procedures or as bio Enterococcus faecium and Clostridioides difficile survival indicators for the control of disinfection processes for on inanimate surfaces for days to months is well known decades. This aspect is also considered in the standard [1, 2]. This potential of persistence results in an increased procedures for testing chemical disinfectants, as outlined risk of acquiring such nosocomial pathogen if a patient in DIN EN 14885:2019 [19]. The phase 3 testing includes is accommodated in a room, which previously was occu- field testing to evaluate the efficacy of new technologies. pied by an infected or colonized patient [3]. In addition, This involves demonstrating performance under the spe - contaminated inanimate surfaces become relevant for the cific local conditions of the users, using naturally occur - transmission of pathogens due to failures in the process ring microorganisms [19]. of surface disinfection and insufficient compliance with The aim of the following study was to compare the hand hygiene [4, 5]. reduction rate of ten different clinical isolates of van - Therefore, automatic room disinfection devices were comycin-resistant enterococci (VRE) with Enterococ- discussed in the last decade as a possible solution to cus hirae ATCC 10541, a common test strain used for improve surface disinfection. Due to the safety and oper- airborne automatic room disinfection procedures. The ational difficulties in using gaseous procedures or nebu - comparison was conducted after exposing the samples to lizers, UV-C disinfection has also been evaluated [6–8]. UV-C radiation, which the manufacturer specified would In the case of manually applied liquid disinfectants, a achieve a 4 log reduction on surfaces. standardized dosage is used, which is usually consistently above the tolerance limits for a wide range of pathogens. Methods The occurring errors in the process of manual room dis - To investigate the differences in sensitivity of various infection are mostly due to the difficulty in reaching each clinical isolates of vancomycin-resistant enterococci surface and ensuring the correct contact time during the (VRE) to UV-C radiation, ceramic tiles contaminated process in the complex hospital environment [9]. Rarely, with ten different VRE were placed at a distance of 1.0 there are also consistent dosing errors of the disinfectant and 1.5  m from the UVD robot model C (UVD Robots, [10]. In contrast, automated room disinfection methods Odense S, Denmark) and irradiated for 20  s. Since the generally do not omit relevant surfaces, but can apply manufacturer specifies an emittance of minimum 2,500 very different, not always predictable, doses of the active µW/cm² at a distance of 1  m, the calculated UV-C irra- ingredient to different surfaces in the room, as recently diation doses were 50 mJ/cm² and 22 mJ/cm² at distances shown for H O nebulizers [11]. of 1.0 and 1.5 m, respectively. The achievement of 50 mJ/ 2 2 UV-C light is known to be an effective disinfectant cm² was confirmed in individual experiments by the against a wide range of microorganisms [12]. But to colour change of a UV-C indicator (UVC Dosimeter™, successfully inactivate pathogens on surfaces, patho- UVD Robots, Odense S, Denmark). gen-dependent germicidal UV-C doses are required. The classical multilocus sequence typing (MLST) However, different values for germicidal activity against sequence types (ST) as well as the core genome MLST the same pathogens are reported in the current literature (cgMLST) cluster types (CT) were determined from [13]. These differences are mainly due to varying condi - a large strain collection of clinical VRE isolates with tions of testing. For example, many UV-C doses for the available whole genome using the SeqSphere software inactivation of pathogens are investigated in air or water, (Ridom, Münster, Germany) [20]. Ten isolates with the which is different compared to the disinfection of dry widest possible genomic distance (different ST and/or surfaces [14, 15]. Furthermore, use of different test organ - CT) were selected for further investigations. The occur - isms and strains of the same pathogen may lead to differ - rence of resistance genes was determined using Res- ent results [15]. Also, different conditions of test rooms, Finder 4.0 [21] and LRE-Finder 1.0 [22, 23]. laboratory or hospital environment, as well as distances For the preparation of standardized contaminated sur- and radiation angle can influence the locally reached faces, suspensions of 1.0–5.0 × 10 colony forming units dose at different room positions and thus the effective - (cfu)/mL were produced for each clinical isolate of VRE. ness of the disinfection process, especially if shadowing is To be representative for realistic protein and dust load of present [16, 17]. surfaces, 0.3% bovine serum albumin (BSA) solution was In this context, current guidelines for using automated used. 20 µL of these suspensions were spread out and room disinfection systems recommend that users con- dried on ceramic tiles (5 × 5  cm, #3709PN00, Villeroy & duct microbiological verification tests in the respective Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 3 of 7 Boch, Mettlach, Germany) resulting in microbial burden and were vortexed for 30  s to elute the recovered bacte- of approx. 10  cfu/25 cm² (suppl. Fig. S1). ria. Afterwards, 100 µL of Aimes medium were spread on The surfaces were positioned at a distance of 1.0 and Columbia agar with 5% Sheep Blood (bioMérieux, Marcy 1.5 m to the UVD robot on a one-meter-high table with l’Etoile, France) to perform quantitative culture in double a black surface in a 6 m³ test room with an anteroom. The determination (detection limit 5 cfu/25 cm²). Subsequently, white wall behind the table was covered by a black sur- the plates were incubated for 18–24 h at 37 °C. Finally, the face, so that the scattered light has minimal influence on colony forming units were determined and the mean value the reduction. Within the irradiation process, the UVD of both approaches were calculated. robot has a 3-minute warm-up phase. To exclude the To calculate the reduction factors, log of the control effect of irradiation during warm-up from the measure - were subtracted from the log of irradiated surfaces. If ment of the reduction, the warm up was performed in the there was no detection of bacteria, the log of the con- anteroom. Therefore, the robot was set up to automati - trol was given as reduction factor, analogue to the specifi - cally enter the test room after warming up in the ante- cations in DIN EN 17272:2020 [24]. room and take up position at a distance of 1 or 1.5  m The differences between the observed reduction fac - from the surfaces (suppl. Fig. S2). tors were statistically analysed by performing one-way For each experiment, four surfaces were positioned ANOVA followed by Tukey post-hoc tests to carry out at both distances. In each case, two of the surfaces were pairwise comparisons of the groups using R (version contaminated with the same VRE isolate. The order of the 4.0.3) and Rstudio (version 2021.09.1) [25]. Therefore, the VRE contaminated surfaces at the distance lines on the R packages tidyverse, ggpubr, rstatix, psych and car were table were changed between the experiments to ensure activated [26–30]. Preliminary tests to check normal- that the collected data were not dependent on the posi- ity assumption (shapiro_test) and variance homogeneity tion. In addition, for each VRE isolate two contaminated (residual vs. fits plot) were performed. A Confidence level surfaces were positioned outside the treated room as a of 95% was used. For all analyses, p-values less than 0.01 control. With this setup, the experiment was performed were considered statistically significant for the compari - five times for each of the ten VRE isolates. son of individual strains, since a significance level of 95% During the irradiation, the relative humidity and tem- was not considered sufficiently meaningful due to natu - perature of the room were measured with LogTag Trex-8 rally occurring variations in microbial growth. (CiK Solutions GmbH, Karlsruhe, Germany) to ensure that these parameters were constant and had no signifi - Results cant influence on the reduction factors. The clinical VRE isolates included in the study showed Following irradiation, bacteria were recovered from between 72 and 428 different alleles in cgMLST (Fig.  1). treated and untreated surfaces. To do this, surfaces were Nine different classical MLST sequence types were repre - wiped off in horizontal, vertical and diagonal directions sented (ST18, ST80, ST78, ST117, ST203, ST262, ST551, using flocked nylon swabs (eSwab™ Standard, Copan; Bres - ST780, ST1283), of which the doubly occurring ST117 cia, Italy). Next, the swabs were placed in Aimes Medium represented two different cluster types (CT71, CT118) as Fig. 1 Minimum spanning tree of the core genome sequences of the investigated isolates. Each circle (node) represents one isolate. The number be- tween the nodes indicates the number of allele differences. The upper numbers in each circle indicate the Sequence type (ST ). The lower numbers indi - cate the Cluster type (CT ), if applicable. White or gray nodes represent isolates carrying VanA or VanB resistance determinants, respectively Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 4 of 7 determined by cgMLST. Five isolates each showed VanA (-0.961, 95% CI (-1.72 to -0.204), p = 0.0029) when 22 mJ/ (ST18, ST78, ST80, ST117/CT118, ST1283) or VanB cm² is applied. (ST117/CT71, ST203, ST262, ST551, ST780) as resis- tance mechanism against glycopeptides, respectively. The Discussion isolate of ST117/CT71 was reported to be phenotypically UV-C irradiation of ten highly divers clinical isolates of van- resistant against linezolid. Using the LRE-Finder resis- comycin-resistant enterococci (VRE) and a common test tance could be genetically confirmed with a wild type/ organism Enterococcus hirae (E. hirae) ATCC 10541 showed mutant ratio of 57.7/42.3% of the mutation G2576T of differences in log reduction between the tested isolates. the 23 S alleles. For the most tolerant isolate only an average log reduction The high contaminated surfaces used for experiments of 3.09 and 2.12 was observed at 50 and 22  mJ/cm² UV-C had an average contamination of 2.3 × 10   cfu/25  cm² irradiation, respectively, in comparison to the most suscep- (suppl. Fig. S1). Measurement of relative humidity and tible one, which showed 4.01 and 3.61 log . The statisti - temperature showed comparative values for all experi- cal analysis revealed significant differences in the observed ments (data not shown). For the UV-C dose of 50  mJ/ reductions between the most tolerant and most susceptible cm² average reductions between 3.09 and 4.27 log were strains. Interestingly, the reduction of clinical isolate ST1283 achieved (Fig.  2). VRE ST1283 and ST80 were observed is significant lower compared to E. hirae ATCC 10541, to be the most UV-C tolerant of the tested clinical iso- when applying UV-C dose of 22 mJ/cm², indicating that this lates, only reduced by means of 3.09 (median 3.10, SD strain is insufficiently representative of particularly tolerant 0.385) and 3.12 (median 2.96, SD 0.63) log . The greatest strains. This is especially important because, in contrast to reduction was observed for ST117/CT118, reduced by wipe disinfection with a standardized concentration of the average 4.27 log (median 4.01, SD 0.82). The common active ingredient, automated room disinfection processes test organism Enterococcus hirae ATCC 10541 showed apply a broad dose spectrum of the respective active prin- the fourth lowest reduction (mean 3.48 log , median ciple to different surfaces in the room. For the linezolid- 3.42, SD 0.304). resistant VRE ST177/CT71, neither an increased nor a For the UV-C dose of 22 mJ/cm², the order of the tested decreased reduction could be observed compared to the clinical isolates slightly changed in terms of the reduction other VRE isolates. Therefore, it can be assumed that line - achieved. ST1283 was observed to be the most tolerant zolid resistance has no effect on UV-C tolerance. isolate with mean reduction of 2.12 log (median 2.15, Current literature indicates that the UV-C dose SD 0.395) followed by ST80 displaying a mean reduction required to inactivate bacteria depends on several factors of 2.41 log (median 2.42 log , SD 0.518). An average such as type of organism, medium and surface area [17]. 10 10 reduction range of 2.12 to 3.61 log was observed. For the inactivation of Enterococcus faecium on solid One-way ANOVA was conducted to asses if the surfaces, the manufacturer specifies a dose of 50  mJ/ observed differences in reduction between the ten clini - cm², which should be reached with an exposure time cal VRE isolates and the common test organism E. hirae of 18.5  s at a distance of 1  m to achieve a reduction of ATCC 10541 were statistically significant. The ANOVA 4 log [31]. However, this value was derived from labo- was statistically significant for both UV-C doses (50  mJ/ ratory experiments. For the experiments carried out in cm²: F(10, 99) = 4.702, p = 1.72 e-05, generalized eta this study, UV-C doses analogous to the manufacturer’s squared = 0.322; 22  mJ/cm²: F(10, 99) = 6.725, p =  6.6 specifications were used and contaminated surfaces were e-08, generalized eta squared = 0.405). Therefore, differ - positioned horizontal but in direct line to the UV-C light ences between the tested groups have to be assumed. source. Under these conditions, the indicated reduction Subsequent Post-hoc Tukey analyses showed signifi - of 4 log was not achieved in either tested E. hirae or cant differences ( p < 0.01) in reduction for 50  mJ/cm² clinical isolates of VRE, although horizontally placed UV between most tolerant isolates ST80 to most susceptible indicators showed the achievement of the desired dose. strain ST117/CT118. In addition, reduction of ST117/ This is consistent with the observation made with other CT118 is significantly different to ST1283 and ST262. methods of room disinfection that, in realistic testing, the For UV-C dose 22  mJ/cm² statistically significant differ - dose to be applied must be higher compared to the doses ences were observed for most tolerant isolate ST1283 of the disinfection product recommended by the manu- to ST780, ST117/CT71, E. hirae, ST203 and ST117/ facturer and defined in the context of approval studies CT118. Furthermore, the difference in reduction of [11]. the most susceptible strain ST117/CT118 to ST80 and Nerandzic et al. observed a mean reduction for clinical ST262 was statistically significant. The detailed results of strains of VRE on benchtop surfaces in a range of approx. 3.2 Tukey analyses are shown in suppl. Table S3. The differ - to 4.4 log using an UV-C dose of 22 mJ/cm² [15]. Mahida ence between the clinical VRE isolates to E. hirae ATCC et al. also reported a mean reduction of clinical VRE strains 10541 is statistically significant only compared to ST1283 seeded on petri dishes greater 4 log applying 22  mJ/cm² 10 Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 5 of 7 Fig. 2 Reduction of different clinical VRE isolates with UV-C doses of 50 (A) and 22 mJ/cm² (B). The boxplots represent the reduction factors determined per tested VRE isolate (n = 10) and test organism E. hirae ATCC 10541 in 5 independent experiments, comparing microbial load on ceramic tiles before and after UV-C radiation with 50 (A) and 22 mJ/cm² (B). Boxplots are in ascending order by median. Statistically significant results (p < 0.01) of pairwise Tukey post-hoc analysis are connected by brackets. Non-significant comparisons are not shown. The p-values are displayed as follows: p < 0.01 = *, p < 0.001 = **, p < 0.0001 = ***, and p < 0.00001 = **** Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 6 of 7 in direct line [32]. Finally, Jelden et al. detected an average it should be noted that there were slight deviations in the reduction range of 3.8 to 4.9 log for vancomycin-resistant disinfection position as part of the robot’s automated travel Enterococcus faecalis ATCC 51299 on different surface process. The resulting variation in the source to surface material, using partially much higher UV-C doses in a range distances could have led to slightly different UV-C doses of mean 9 to 688 mJ/cm² [17]. In our study, we observed a between the experiments. Furthermore, it has to be taken mean reduction range of 2.12 to 3.61 log for the ten inves- into account that the experiments were conducted with very tigated VRE isolates when using the direct radiation dose of high surface contaminations in order to enable calculation 22 mJ/cm² to disinfect the contaminated ceramic tiles. The of reduction efficacy over a range of several log reduction. observed reductions were up to 1 log lower than the given However, realistic contamination of surfaces, even in hospi- literature values. tal environments, is much lower [33, 34], so that the disin- Nerandzic et al. studied three different clinical VRE fection procedure could be effective even if the bactericidal strains, of which two were VanA and one VanB positive iso- efficacy is not reached. Finally, despite the use of the most lates. The obtained results indicated differences in reduction diverse VRE isolates as possible, it cannot be excluded that rates of these. While the Van B VRE isolate was reduced just isolates with even higher tolerance to UV-C radiation exist above 3 log , the Van A isolates showed reduction from in clinical practice. approx. 3.75 up to just under 4.5 log [15]. The observed differences between the most tolerant and the most suscep - Conclusion tible VRE isolate are in a range of > 1 log . A comparable Since manual cleaning and disinfection are frequently inad- range was determined in the present study. However, no equate to remove pathogens on surfaces completely, auto- correlation of more or less susceptibility depending on resis- matic room decontamination with UV-C radiation could tance mechanism against glycopeptides could be observed. be a suitable method to increase efficacy of surface disinfec - The strain with genetically confirmed resistance against gly - tion. However, before using it routinely, efficacy verification copeptides displayed no significant differences compared to is recommended. Testing the efficacy of UV-C radiation the laboratory strain E. hirae ATCC 10541, whereas a signif- for ten different whole-genome-sequenced clinical isolates icantly higher reduction was observed for the low radiation of VRE as well as ATCC 10541 test organism Enterococ- dose compared to the most tolerant isolate. This data indi - cus hirae, showed differences in reduction. Use of UV-C cates that mutations in the 23 S alleles do not interfere with radiation according to manufacturer’s instructions did not the behavior towards UV-C radiation. Both previous data achieve the proposed reduction. In addition, some clinical and the results obtained in this study support the recom- isolates of VRE are more stable against UV-C radiation than mendation of Beswick et al. to conduct tests in the intended the common test organism. Therefore, to establish the use clinical environment to verify the efficacy of disinfection of such devices, additional efficacy tests in hospital environ - methods [18]. This is particularly crucial because manu - ments using clinical isolates should be performed. facturers’ specifications may not provide sufficient guid - ance to achieve bactericidal efficacy against clinical isolates, Supplementary Information The online version contains supplementary material available at https://doi. which may exhibit higher tolerance than the test organism. org/10.1186/s13756-023-01259-3. Moreover, variations in room conditions, surface materi- als and shadowing can influence the required UV-C dose Supplementary Material 1 for effective disinfection, as evidenced by the previously Supplementary Material 2 described differences in reduction at the same UV-C dose. Supplementary Material 3 The standard procedure for testing chemical disinfectants, as defined by DIN EN 14885:2019, stipulates a consistent Acknowledgements concentration of the active ingredient to guarantee effec - We thank Rieke Hinrichs for excellent technical assistance. We acknowledge tive disinfection. However, determining a minimum efficacy financial support from the Open Access Publication Fund of UKE - dose for UV-C is not as straightforward. Consequently, it is Universitätsklinikum Hamburg-Eppendorf and DFG – German Research Foundation. advisable to conduct a phase 3 test for automated UV-C dis- infection processes to validate their efficacy in routine use Author contributions across diverse hospital settings. In this regard, more tolerant JK and BK conceived and planned the experiment, in consultation with MC. BK and JK carried out the experiments. GF, JK, EK, HB, CBC, PM and BK analysed clinical isolates should be used in the future. the data and interpreted the results. BK performed the statistical analysis of The following limitations of the present study should be the data. BK and JK prepared the original draft of manuscript in consultation noted: Firstly, the experiments were performed with only with GF. CBC, EK, MC, HB, PM and GF substantively revised the manuscript. All authors read and approved the final manuscript. one standard material. Since several studies show differ - ences depending on surface material (e.g. [17]), further Funding experiments should be performed with other surface mate- Open Access funding enabled and organized by Projekt DEAL. UVD-Robots enables a free exchange of UVD robot model B to model C. rials frequently used in the clinical environment. In addition, Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 7 of 7 Data Availability 14. McKinney CW, Pruden A. Ultraviolet disinfection of antibiotic resistant bacte- The datasets used and/or analysed during this study are included in this ria and their antibiotic resistance genes in water and wastewater. 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Sensitivity of Bacteria, Protozoa, Viruses, and other microorganisms to Ultraviolet Radiation. J RES NATL INST STAN. Publisher’s Note 2021. https://doi.org/10.6028/jres.126.021. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antimicrobial Resistance and Infection Control Springer Journals

Tolerance of clinical vancomycin-resistant Enterococcus faecium isolates against UV-C light from a mobile source

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Abstract

Background Admission to a room previously occupied by patients carrying environmentally robust pathogens implies an increased risk of acquiring those pathogens. Therefore, ‘No-touch’ automated room disinfection systems, including devices based on UV-C irradiation, are discussed to improve terminal cleaning. It is still unclear if clinical isolates of relevant pathogens behave differently under UV-C irradiation compared to laboratory strains used in the approval process of disinfection procedures. In this study we analysed the susceptibility of well characterized clonally divergent vancomycin-resistant enterococci ( VRE) strains, including a linezolid-resistant isolate, against UV-C radiation. Methods Susceptibility against UV-C of ten clonally divergent clinical isolates of VRE was determined in comparison to the commonly used test organism Enterococcus hirae ATCC 10541. Ceramic tiles contaminated with 10 to 10 colony forming units/25 cm² of the different enterococci were positioned at a distance of 1.0 and 1.5 m and irradiated for 20 s, resulting in a UV-C dose of 50 and 22 mJ/cm², respectively. Reduction factors were calculated after quantitative culture of the bacteria recovered from treated and untreated surfaces. Results Susceptibility to UV-C varied considerably among the strains studied, with the mean value of the most robust strain being up to a power of ten lower compared to the most sensitive strain at both UV-C doses. The two most tolerant strains belonged to MLST sequence types ST80 and ST1283. The susceptibility of the laboratory strain E. hirae ATCC 10541 ranged between the most sensitive and most tolerant isolates for both irradiation doses. However, for UV-C dose of 22 mJ/cm², the reduction of the most tolerant isolate of ST1283 was statistically significantly lower compared to E. hirae ATCC 10541. The most susceptible strains belonged to the MLST sequence types ST117 and ST203. Conclusions These results indicate that UV-C doses reported in the literature are sufficient for the reduction of commonly used reference strains of enterococci but could be insufficient for the reduction of tolerant patient VRE- isolates in a hospital setting. Therefore, for future studies, the most tolerant clinical isolates should be used to validate automated UV-C devices or longer exposure times should be expected to ensure efficacy in the real world. Keywords Automatic room decontamination, Disinfection, UV-C, UV-irradiation, vancomycin resistant enterococci *Correspondence: Institute for Medical Microbiology, Virology and Hygiene, Department J. K. Knobloch Infection Prevention and Control, University Medical Center Hamburg- j.knobloch@uke.de Eppendorf, Hamburg, Germany Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 2 of 7 Background clinical environment to confirm efficacy [ 18]. For these In recent decades, contamination of surfaces in the efficacy tests, it is important to consider that recent clini - patient environment as cause for acquisition of noso- cal isolates may behave differently compared to labo - comial infections and transmission of pathogens has ratory strains, which have been commonly used in the gained importance. Especially for Staphylococcus aureus, approval process of disinfection procedures or as bio Enterococcus faecium and Clostridioides difficile survival indicators for the control of disinfection processes for on inanimate surfaces for days to months is well known decades. This aspect is also considered in the standard [1, 2]. This potential of persistence results in an increased procedures for testing chemical disinfectants, as outlined risk of acquiring such nosocomial pathogen if a patient in DIN EN 14885:2019 [19]. The phase 3 testing includes is accommodated in a room, which previously was occu- field testing to evaluate the efficacy of new technologies. pied by an infected or colonized patient [3]. In addition, This involves demonstrating performance under the spe - contaminated inanimate surfaces become relevant for the cific local conditions of the users, using naturally occur - transmission of pathogens due to failures in the process ring microorganisms [19]. of surface disinfection and insufficient compliance with The aim of the following study was to compare the hand hygiene [4, 5]. reduction rate of ten different clinical isolates of van - Therefore, automatic room disinfection devices were comycin-resistant enterococci (VRE) with Enterococ- discussed in the last decade as a possible solution to cus hirae ATCC 10541, a common test strain used for improve surface disinfection. Due to the safety and oper- airborne automatic room disinfection procedures. The ational difficulties in using gaseous procedures or nebu - comparison was conducted after exposing the samples to lizers, UV-C disinfection has also been evaluated [6–8]. UV-C radiation, which the manufacturer specified would In the case of manually applied liquid disinfectants, a achieve a 4 log reduction on surfaces. standardized dosage is used, which is usually consistently above the tolerance limits for a wide range of pathogens. Methods The occurring errors in the process of manual room dis - To investigate the differences in sensitivity of various infection are mostly due to the difficulty in reaching each clinical isolates of vancomycin-resistant enterococci surface and ensuring the correct contact time during the (VRE) to UV-C radiation, ceramic tiles contaminated process in the complex hospital environment [9]. Rarely, with ten different VRE were placed at a distance of 1.0 there are also consistent dosing errors of the disinfectant and 1.5  m from the UVD robot model C (UVD Robots, [10]. In contrast, automated room disinfection methods Odense S, Denmark) and irradiated for 20  s. Since the generally do not omit relevant surfaces, but can apply manufacturer specifies an emittance of minimum 2,500 very different, not always predictable, doses of the active µW/cm² at a distance of 1  m, the calculated UV-C irra- ingredient to different surfaces in the room, as recently diation doses were 50 mJ/cm² and 22 mJ/cm² at distances shown for H O nebulizers [11]. of 1.0 and 1.5 m, respectively. The achievement of 50 mJ/ 2 2 UV-C light is known to be an effective disinfectant cm² was confirmed in individual experiments by the against a wide range of microorganisms [12]. But to colour change of a UV-C indicator (UVC Dosimeter™, successfully inactivate pathogens on surfaces, patho- UVD Robots, Odense S, Denmark). gen-dependent germicidal UV-C doses are required. The classical multilocus sequence typing (MLST) However, different values for germicidal activity against sequence types (ST) as well as the core genome MLST the same pathogens are reported in the current literature (cgMLST) cluster types (CT) were determined from [13]. These differences are mainly due to varying condi - a large strain collection of clinical VRE isolates with tions of testing. For example, many UV-C doses for the available whole genome using the SeqSphere software inactivation of pathogens are investigated in air or water, (Ridom, Münster, Germany) [20]. Ten isolates with the which is different compared to the disinfection of dry widest possible genomic distance (different ST and/or surfaces [14, 15]. Furthermore, use of different test organ - CT) were selected for further investigations. The occur - isms and strains of the same pathogen may lead to differ - rence of resistance genes was determined using Res- ent results [15]. Also, different conditions of test rooms, Finder 4.0 [21] and LRE-Finder 1.0 [22, 23]. laboratory or hospital environment, as well as distances For the preparation of standardized contaminated sur- and radiation angle can influence the locally reached faces, suspensions of 1.0–5.0 × 10 colony forming units dose at different room positions and thus the effective - (cfu)/mL were produced for each clinical isolate of VRE. ness of the disinfection process, especially if shadowing is To be representative for realistic protein and dust load of present [16, 17]. surfaces, 0.3% bovine serum albumin (BSA) solution was In this context, current guidelines for using automated used. 20 µL of these suspensions were spread out and room disinfection systems recommend that users con- dried on ceramic tiles (5 × 5  cm, #3709PN00, Villeroy & duct microbiological verification tests in the respective Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 3 of 7 Boch, Mettlach, Germany) resulting in microbial burden and were vortexed for 30  s to elute the recovered bacte- of approx. 10  cfu/25 cm² (suppl. Fig. S1). ria. Afterwards, 100 µL of Aimes medium were spread on The surfaces were positioned at a distance of 1.0 and Columbia agar with 5% Sheep Blood (bioMérieux, Marcy 1.5 m to the UVD robot on a one-meter-high table with l’Etoile, France) to perform quantitative culture in double a black surface in a 6 m³ test room with an anteroom. The determination (detection limit 5 cfu/25 cm²). Subsequently, white wall behind the table was covered by a black sur- the plates were incubated for 18–24 h at 37 °C. Finally, the face, so that the scattered light has minimal influence on colony forming units were determined and the mean value the reduction. Within the irradiation process, the UVD of both approaches were calculated. robot has a 3-minute warm-up phase. To exclude the To calculate the reduction factors, log of the control effect of irradiation during warm-up from the measure - were subtracted from the log of irradiated surfaces. If ment of the reduction, the warm up was performed in the there was no detection of bacteria, the log of the con- anteroom. Therefore, the robot was set up to automati - trol was given as reduction factor, analogue to the specifi - cally enter the test room after warming up in the ante- cations in DIN EN 17272:2020 [24]. room and take up position at a distance of 1 or 1.5  m The differences between the observed reduction fac - from the surfaces (suppl. Fig. S2). tors were statistically analysed by performing one-way For each experiment, four surfaces were positioned ANOVA followed by Tukey post-hoc tests to carry out at both distances. In each case, two of the surfaces were pairwise comparisons of the groups using R (version contaminated with the same VRE isolate. The order of the 4.0.3) and Rstudio (version 2021.09.1) [25]. Therefore, the VRE contaminated surfaces at the distance lines on the R packages tidyverse, ggpubr, rstatix, psych and car were table were changed between the experiments to ensure activated [26–30]. Preliminary tests to check normal- that the collected data were not dependent on the posi- ity assumption (shapiro_test) and variance homogeneity tion. In addition, for each VRE isolate two contaminated (residual vs. fits plot) were performed. A Confidence level surfaces were positioned outside the treated room as a of 95% was used. For all analyses, p-values less than 0.01 control. With this setup, the experiment was performed were considered statistically significant for the compari - five times for each of the ten VRE isolates. son of individual strains, since a significance level of 95% During the irradiation, the relative humidity and tem- was not considered sufficiently meaningful due to natu - perature of the room were measured with LogTag Trex-8 rally occurring variations in microbial growth. (CiK Solutions GmbH, Karlsruhe, Germany) to ensure that these parameters were constant and had no signifi - Results cant influence on the reduction factors. The clinical VRE isolates included in the study showed Following irradiation, bacteria were recovered from between 72 and 428 different alleles in cgMLST (Fig.  1). treated and untreated surfaces. To do this, surfaces were Nine different classical MLST sequence types were repre - wiped off in horizontal, vertical and diagonal directions sented (ST18, ST80, ST78, ST117, ST203, ST262, ST551, using flocked nylon swabs (eSwab™ Standard, Copan; Bres - ST780, ST1283), of which the doubly occurring ST117 cia, Italy). Next, the swabs were placed in Aimes Medium represented two different cluster types (CT71, CT118) as Fig. 1 Minimum spanning tree of the core genome sequences of the investigated isolates. Each circle (node) represents one isolate. The number be- tween the nodes indicates the number of allele differences. The upper numbers in each circle indicate the Sequence type (ST ). The lower numbers indi - cate the Cluster type (CT ), if applicable. White or gray nodes represent isolates carrying VanA or VanB resistance determinants, respectively Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 4 of 7 determined by cgMLST. Five isolates each showed VanA (-0.961, 95% CI (-1.72 to -0.204), p = 0.0029) when 22 mJ/ (ST18, ST78, ST80, ST117/CT118, ST1283) or VanB cm² is applied. (ST117/CT71, ST203, ST262, ST551, ST780) as resis- tance mechanism against glycopeptides, respectively. The Discussion isolate of ST117/CT71 was reported to be phenotypically UV-C irradiation of ten highly divers clinical isolates of van- resistant against linezolid. Using the LRE-Finder resis- comycin-resistant enterococci (VRE) and a common test tance could be genetically confirmed with a wild type/ organism Enterococcus hirae (E. hirae) ATCC 10541 showed mutant ratio of 57.7/42.3% of the mutation G2576T of differences in log reduction between the tested isolates. the 23 S alleles. For the most tolerant isolate only an average log reduction The high contaminated surfaces used for experiments of 3.09 and 2.12 was observed at 50 and 22  mJ/cm² UV-C had an average contamination of 2.3 × 10   cfu/25  cm² irradiation, respectively, in comparison to the most suscep- (suppl. Fig. S1). Measurement of relative humidity and tible one, which showed 4.01 and 3.61 log . The statisti - temperature showed comparative values for all experi- cal analysis revealed significant differences in the observed ments (data not shown). For the UV-C dose of 50  mJ/ reductions between the most tolerant and most susceptible cm² average reductions between 3.09 and 4.27 log were strains. Interestingly, the reduction of clinical isolate ST1283 achieved (Fig.  2). VRE ST1283 and ST80 were observed is significant lower compared to E. hirae ATCC 10541, to be the most UV-C tolerant of the tested clinical iso- when applying UV-C dose of 22 mJ/cm², indicating that this lates, only reduced by means of 3.09 (median 3.10, SD strain is insufficiently representative of particularly tolerant 0.385) and 3.12 (median 2.96, SD 0.63) log . The greatest strains. This is especially important because, in contrast to reduction was observed for ST117/CT118, reduced by wipe disinfection with a standardized concentration of the average 4.27 log (median 4.01, SD 0.82). The common active ingredient, automated room disinfection processes test organism Enterococcus hirae ATCC 10541 showed apply a broad dose spectrum of the respective active prin- the fourth lowest reduction (mean 3.48 log , median ciple to different surfaces in the room. For the linezolid- 3.42, SD 0.304). resistant VRE ST177/CT71, neither an increased nor a For the UV-C dose of 22 mJ/cm², the order of the tested decreased reduction could be observed compared to the clinical isolates slightly changed in terms of the reduction other VRE isolates. Therefore, it can be assumed that line - achieved. ST1283 was observed to be the most tolerant zolid resistance has no effect on UV-C tolerance. isolate with mean reduction of 2.12 log (median 2.15, Current literature indicates that the UV-C dose SD 0.395) followed by ST80 displaying a mean reduction required to inactivate bacteria depends on several factors of 2.41 log (median 2.42 log , SD 0.518). An average such as type of organism, medium and surface area [17]. 10 10 reduction range of 2.12 to 3.61 log was observed. For the inactivation of Enterococcus faecium on solid One-way ANOVA was conducted to asses if the surfaces, the manufacturer specifies a dose of 50  mJ/ observed differences in reduction between the ten clini - cm², which should be reached with an exposure time cal VRE isolates and the common test organism E. hirae of 18.5  s at a distance of 1  m to achieve a reduction of ATCC 10541 were statistically significant. The ANOVA 4 log [31]. However, this value was derived from labo- was statistically significant for both UV-C doses (50  mJ/ ratory experiments. For the experiments carried out in cm²: F(10, 99) = 4.702, p = 1.72 e-05, generalized eta this study, UV-C doses analogous to the manufacturer’s squared = 0.322; 22  mJ/cm²: F(10, 99) = 6.725, p =  6.6 specifications were used and contaminated surfaces were e-08, generalized eta squared = 0.405). Therefore, differ - positioned horizontal but in direct line to the UV-C light ences between the tested groups have to be assumed. source. Under these conditions, the indicated reduction Subsequent Post-hoc Tukey analyses showed signifi - of 4 log was not achieved in either tested E. hirae or cant differences ( p < 0.01) in reduction for 50  mJ/cm² clinical isolates of VRE, although horizontally placed UV between most tolerant isolates ST80 to most susceptible indicators showed the achievement of the desired dose. strain ST117/CT118. In addition, reduction of ST117/ This is consistent with the observation made with other CT118 is significantly different to ST1283 and ST262. methods of room disinfection that, in realistic testing, the For UV-C dose 22  mJ/cm² statistically significant differ - dose to be applied must be higher compared to the doses ences were observed for most tolerant isolate ST1283 of the disinfection product recommended by the manu- to ST780, ST117/CT71, E. hirae, ST203 and ST117/ facturer and defined in the context of approval studies CT118. Furthermore, the difference in reduction of [11]. the most susceptible strain ST117/CT118 to ST80 and Nerandzic et al. observed a mean reduction for clinical ST262 was statistically significant. The detailed results of strains of VRE on benchtop surfaces in a range of approx. 3.2 Tukey analyses are shown in suppl. Table S3. The differ - to 4.4 log using an UV-C dose of 22 mJ/cm² [15]. Mahida ence between the clinical VRE isolates to E. hirae ATCC et al. also reported a mean reduction of clinical VRE strains 10541 is statistically significant only compared to ST1283 seeded on petri dishes greater 4 log applying 22  mJ/cm² 10 Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 5 of 7 Fig. 2 Reduction of different clinical VRE isolates with UV-C doses of 50 (A) and 22 mJ/cm² (B). The boxplots represent the reduction factors determined per tested VRE isolate (n = 10) and test organism E. hirae ATCC 10541 in 5 independent experiments, comparing microbial load on ceramic tiles before and after UV-C radiation with 50 (A) and 22 mJ/cm² (B). Boxplots are in ascending order by median. Statistically significant results (p < 0.01) of pairwise Tukey post-hoc analysis are connected by brackets. Non-significant comparisons are not shown. The p-values are displayed as follows: p < 0.01 = *, p < 0.001 = **, p < 0.0001 = ***, and p < 0.00001 = **** Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 6 of 7 in direct line [32]. Finally, Jelden et al. detected an average it should be noted that there were slight deviations in the reduction range of 3.8 to 4.9 log for vancomycin-resistant disinfection position as part of the robot’s automated travel Enterococcus faecalis ATCC 51299 on different surface process. The resulting variation in the source to surface material, using partially much higher UV-C doses in a range distances could have led to slightly different UV-C doses of mean 9 to 688 mJ/cm² [17]. In our study, we observed a between the experiments. Furthermore, it has to be taken mean reduction range of 2.12 to 3.61 log for the ten inves- into account that the experiments were conducted with very tigated VRE isolates when using the direct radiation dose of high surface contaminations in order to enable calculation 22 mJ/cm² to disinfect the contaminated ceramic tiles. The of reduction efficacy over a range of several log reduction. observed reductions were up to 1 log lower than the given However, realistic contamination of surfaces, even in hospi- literature values. tal environments, is much lower [33, 34], so that the disin- Nerandzic et al. studied three different clinical VRE fection procedure could be effective even if the bactericidal strains, of which two were VanA and one VanB positive iso- efficacy is not reached. Finally, despite the use of the most lates. The obtained results indicated differences in reduction diverse VRE isolates as possible, it cannot be excluded that rates of these. While the Van B VRE isolate was reduced just isolates with even higher tolerance to UV-C radiation exist above 3 log , the Van A isolates showed reduction from in clinical practice. approx. 3.75 up to just under 4.5 log [15]. The observed differences between the most tolerant and the most suscep - Conclusion tible VRE isolate are in a range of > 1 log . A comparable Since manual cleaning and disinfection are frequently inad- range was determined in the present study. However, no equate to remove pathogens on surfaces completely, auto- correlation of more or less susceptibility depending on resis- matic room decontamination with UV-C radiation could tance mechanism against glycopeptides could be observed. be a suitable method to increase efficacy of surface disinfec - The strain with genetically confirmed resistance against gly - tion. However, before using it routinely, efficacy verification copeptides displayed no significant differences compared to is recommended. Testing the efficacy of UV-C radiation the laboratory strain E. hirae ATCC 10541, whereas a signif- for ten different whole-genome-sequenced clinical isolates icantly higher reduction was observed for the low radiation of VRE as well as ATCC 10541 test organism Enterococ- dose compared to the most tolerant isolate. This data indi - cus hirae, showed differences in reduction. Use of UV-C cates that mutations in the 23 S alleles do not interfere with radiation according to manufacturer’s instructions did not the behavior towards UV-C radiation. Both previous data achieve the proposed reduction. In addition, some clinical and the results obtained in this study support the recom- isolates of VRE are more stable against UV-C radiation than mendation of Beswick et al. to conduct tests in the intended the common test organism. Therefore, to establish the use clinical environment to verify the efficacy of disinfection of such devices, additional efficacy tests in hospital environ - methods [18]. This is particularly crucial because manu - ments using clinical isolates should be performed. facturers’ specifications may not provide sufficient guid - ance to achieve bactericidal efficacy against clinical isolates, Supplementary Information The online version contains supplementary material available at https://doi. which may exhibit higher tolerance than the test organism. org/10.1186/s13756-023-01259-3. Moreover, variations in room conditions, surface materi- als and shadowing can influence the required UV-C dose Supplementary Material 1 for effective disinfection, as evidenced by the previously Supplementary Material 2 described differences in reduction at the same UV-C dose. Supplementary Material 3 The standard procedure for testing chemical disinfectants, as defined by DIN EN 14885:2019, stipulates a consistent Acknowledgements concentration of the active ingredient to guarantee effec - We thank Rieke Hinrichs for excellent technical assistance. We acknowledge tive disinfection. However, determining a minimum efficacy financial support from the Open Access Publication Fund of UKE - dose for UV-C is not as straightforward. Consequently, it is Universitätsklinikum Hamburg-Eppendorf and DFG – German Research Foundation. advisable to conduct a phase 3 test for automated UV-C dis- infection processes to validate their efficacy in routine use Author contributions across diverse hospital settings. In this regard, more tolerant JK and BK conceived and planned the experiment, in consultation with MC. BK and JK carried out the experiments. GF, JK, EK, HB, CBC, PM and BK analysed clinical isolates should be used in the future. the data and interpreted the results. BK performed the statistical analysis of The following limitations of the present study should be the data. BK and JK prepared the original draft of manuscript in consultation noted: Firstly, the experiments were performed with only with GF. CBC, EK, MC, HB, PM and GF substantively revised the manuscript. All authors read and approved the final manuscript. one standard material. Since several studies show differ - ences depending on surface material (e.g. [17]), further Funding experiments should be performed with other surface mate- Open Access funding enabled and organized by Projekt DEAL. UVD-Robots enables a free exchange of UVD robot model B to model C. rials frequently used in the clinical environment. In addition, Knobling et al. Antimicrobial Resistance & Infection Control (2023) 12:63 Page 7 of 7 Data Availability 14. McKinney CW, Pruden A. Ultraviolet disinfection of antibiotic resistant bacte- The datasets used and/or analysed during this study are included in this ria and their antibiotic resistance genes in water and wastewater. 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Journal

Antimicrobial Resistance and Infection Control – Springer Journals

Published: Jul 4, 2023

Keywords: Automatic room decontamination; Disinfection; UV-C; UV-irradiation; vancomycin resistant enterococci

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