Development of a highly-sensitive method to detect the carriage of carbapenem-resistantPseudomonas aeruginosain humans

18 Carbapenem-resistant Pseudomonas aeruginosa (CRPA) causes severe and potentially life-threatening 19 infections in hospitalized patients with mortality rates of more than 40%. To detect CRPA carriage in 20 humans for surveillance purposes or to prevent spread and outbreaks in hospitals, a highly-sensitive 21 culture method for CRPA carriage in humans is needed. We aimed to develop such a highly-sensitive 22 method, that would be feasible in laboratories with limited resources. In this study, seven well-defined 23 CRPA strains belonging to high-risk clones were used, including one CRPA without a carbapenemase 24 gene and six carbapenem-resistant isolates with carbapenemase genes. We applied a stepwise approach 25 wherein we included four enrichment broths and eight Pseudomonas aeruginosa-selective culture 26 media. Spiking experiments were performed to further evaluate the combination of the most sensitive 27 enrichment broths and selective agar plates in human samples. The two most sensitive enrichments 28 broths were TSB-vancomycin and TSB-vancomycin with 2 mg/L imipenem and the most sensitive 29 selective agar plates were Pseudomonas isolation agar Becton Dickinson, Pseudomonas isolation agar 30 Sigma-Aldrich, and M-PA-C (Becton Dickinson). After the spiking experiment, the best method for 31 detecting CRPA based on the sensitivity and the selectivity was the combination of TSB-vancomycin with 32 2 mg/L imipenem as an enrichment broth for overnight incubation, followed by subculturing the broth 33 on M-PA-C agar plate. We have thus developed a highly-sensitive selective method to detect CRPA 34 carriage in humans, which can also be applied in limited-resource laboratories. This may contribute to 35 an overall effort to control CRPA. 36 37

38 Pseudomonas aeruginosa, carbapenems, drug resistance, culture techniques, culture media, 39 humans 40 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 3

41 Pseudomonas aeruginosa is a Gram-negative bacterium that causes severe and potentially life-42 threatening infections in hospitalized patients (1). The worldwide emergence of carbapenem-resistant P. 43 aeruginosa (CRPA) makes infections by these pathogens almost untreatable, resulting in crude mortality 44 rates of more than 40% (2, 3). The World Health Organization has, consequently, ranked CRPA as “high 45 priority” bacterial pathogen for further action (4). 46 In the hospital setting, actions should be focused on the prevention of transmission of CRPA. 47 Several studies have reported colonization with CRPA in admitted patients, which poses a risk of 48 transmitting these pathogens to other patients or environmental reservoirs where these bacteria may 49 be difficult to eradicate and lead to outbreaks (2, 5, 6). During outbreaks, contact investigations should 50 be performed to identify undetected carriers (7). For these purposes, a highly-sensitive culture method 51 for CRPA carriage in humans is needed. Retrieving viable isolates is essential for antimicrobial 52 susceptibility testing and, if available, analysis of genetic relatedness among isolates (8, 9). 53 In a recent review on this topic (10), a lack of knowledge on the methods to be used for the rapid 54 and sensitive detection of CRPA was revealed, which was reflected by only a few diagnostic accuracy 55 studies comparing different culture methods and a large variety of culture methods described in recent 56 outbreak-surveillance studies. It was suggested, however, that the use of an enrichment broth prior to 57 plating the material on a selective medium would be of benefit, although this was based on only one 58 study. Therefore, the aim of this study was to develop a highly-sensitive culture method for the 59 detection of CRPA carriage in humans. To that end, various enrichment broths (i.e., tryptic soy broth 60 [TSB] with addition of various antibiotics) and P. aeruginosa-selective agar plates were compared, 61 followed by spiking experiments to determine the most sensitive combination of broth and plate to 62 detect CRPA. We aimed to develop a method that would be feasible in laboratories with limited 63 resources as well, thus without application of a nucleic acid amplification technology. 64 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 4

65 General approach 66 The study was performed in the framework of the SAMPAN study ( A Smart Surveillance Strategy for 67 Carbapenem-resistant Pseudomonas aeruginosa) (11). We developed the method in a stepwise manner, 68 by 69 • Testing the growth of well-characterized CRPA strains inoculated into an enrichment broth 70 continued by culturing onto blood agar 71 • Testing the phenotype and growth of well-characterized CRPA strains on various P. aeruginosa 72 selective agar plates 73 • Evaluating the growth of well-characterized CRPA strains from faecal samples spiked with these 74 strains while using the most sensitive enrichment broth from previous experiments continued 75 by culturing onto the most sensitive selective agar plates 76 Bacterial isolates 77 In this study, seven well-defined CRPA strains from Indonesia and the Netherlands were used, 78 including one CRPA without a carbapenemase gene and six carbapenem-resistant isolates with 79 carbapenemase genes, including blaVIM (n=3), blaGES (n=1), blaIMP (n=1), and blaNDM (n=1) (Table 1). 80 Species identification was performed using the Matrix-Assisted Laser Desorption/Ionization Time-Of-81 Flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany). Antibiotic 82 susceptibility was determined by VITEK2® (bioMérieux, Marcy l’Etoile, France) (12). Carbapenem-83 resistance was defined as resistance to at least one of the carbapenems (i.e., imipenem, or 84 meropenem). The results of the susceptibility test were interpreted according to the breakpoints 85 defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (13, 14). Multiplex 86 real-time PCR was performed to detect resistance genes, followed by sequencing to genetically 87 characterize the strains. To make series of bacterial suspensions, inoculums of 0.5 McFarland standard 88 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 5 suspensions (approximately 1.5 × 108 CFU/mL) from each of the CRPA strains were prepared using 0.45% 89 saline. Eight times 10-fold serial dilution from each inoculum suspension was made in a 0.45% sterile 90 saline solution creating bacterial suspensions with different concentration from 1.5 × 108CFU/mL until 91 1.5 CFU/mL. To confirm the purity of the suspensions, 100 µL of the suspensions were inoculated onto 92 tryptic soy agar with 5% sheep blood (i.e., blood agar) (Becton Dickinson Diagnostics, Breda, The 93 Netherlands), followed by spreading the inoculum evenly over the surface of the plate using sterile 94 disposable spatula. The plates were observed the next day to ensure purity and for counting. 95 96 Table 1 Carbapenem-resistant Pseudomonas aeruginosa strains used in this study 97 Sequence type Carbapenem- resistant gene Minimum Inhibitory Concentration (mg/L) Imipenem Meropenem Ceftazidime Strain 11 ST446 blaVIM-2 >=16 (R) 16 (R) 16 (R) Strain 22 ST773 blaNDM >32 (R) >32 (R) >16 (R) Strain 33 ST111 blaVIM-2 > 8 (R) 4 (I) >=32 (R) Strain 44 ST253 blaVIM-2 >=16 (R) >=16 (R) >=16 (R) Strain 55 ST357 blaIMP-7 >=16 (R) >=16 (R) >=32 (R) Strain 65 ST235 blaGES-5 >=16 (R) >=16 (R) 16 (R) Strain 75 ST446 None >=16 (R) >=16 (R) 8 (S) 1Previously published by Van der Zee et al. (15) 98 2From a patient that was hospitalized in Marocco, and was screened after being transferred to the 99 Netherlands. 100 3Previously published by Pirzadian et al. (16) 101 4Strain from a sink drain in the intensive care, previously published by Pirzadian et al. (17) 102 5Previously published by Pelegrin et al. (18) 103 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 6 Comparison of enrichment broths 104 Four enrichments broths, all based on tryptic soy broth (TSB) (Becton Dickinson Diagnostics, Breda, 105 The Netherlands) were included in this comparison (Figure 1): TSB supplemented with 2 mg/L of 106 vancomycin (i.e., TSB-vancomycin), TSB-vancomycin supplemented with 2 mg/L and 4 mg/L of 107 imipenem, and TSB-vancomycin with 6 mg/L of ceftazidime. Vancomycin was added in all broths, as this 108 inhibits Gram-positive bacteria that are present in perianal, rectal swabs or faeces, which are the most 109 used screening samples (6, 10). As a carbapenem antibiotic, imipenem was used because imipenem is 110 more stable than meropenem when using discs to prepare the broth (19). Ceftazidime was chosen 111 based on the general finding that most CRPA are also less susceptible to ceftazidime, and previous 112 experiences (6). The enrichment broths were prepared by adding antibiotic discs to the broth. For 113 instance, to attain 4 mg/L of vancomycin, two discs of 5 µg (Oxoid) were added to 5mL broth. To 114 compare the enrichment broths, 100 µL of the bacterial suspension dilutions were inoculated into the 115 broths. Subsequently, each broth was incubated at 35±1°C for 24 hours, followed by observation of 116 turbidity. After that, regardless of the turbidity, 10 µL of the broth was sub-cultured onto a blood agar 117 plate, which was incubated at 35±1°C for 24 and 48 hours. Blood agar plates were observed for bacterial 118 growth and each plate was scored as either positive (growth) or negative (no growth). All experiments 119 were performed in triplicate. The sensitivity was calculated as the number of positive plates divided by 120 the total number of samples tested per type of broth. 121 122 Comparison of selective agar plates 123 Eight Pseudomonas aeruginosa-selective culture media were tested: ChromID® Pseudomonas 124 aeruginosa (bioMérieux), Pseudosel (cetrimide) agar (bioMérieux), Thermo Scientific™ Pseudomonas C-125 N Selective Agar (Oxoid, Basingstoke, UK), Cetrimide agar (bioMérieux), Pseudomonas isolation agar 126 (PIA; Becton Dickinson Diagnostics, Breda, The Netherlands), PIA (Sigma-Aldrich, St. Louis, MO, USA), M-127 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 7 PA-C agar (Becton Dickinson Diagnostics, Breda, The Netherlands), and Phenanthroline agar (Mueller 128 Hinton, Oxoid, Basingstoke, UK with 50 µg/L 1,10-Phenanthroline, Sigma-Aldrich, St. Louis, MO, USA) 129 (20, 21). Blood agar plates were used as a control (Figure 1). 130 Bacterial suspensions (100 µL each) were inoculated directly onto each agar plate. The dilution was 131 spread evenly over the surface of the agar plates using a disposable spreader. The agar plates were 132 incubated aerobically at 35±1°C, followed by observation of colony characteristics after 18, 24, 42, 48, 133 and 72 hours of incubation. Colony growth on the selective plates was recorded as growth (positive) or 134 no growth (negative). Atypical colonies observed on the plates were identified by MALDI-TOF MS to 135 exclude contamination. The experiment was performed in triplicate. The sensitivity was measured as the 136 number of strains with growth divided by the number of strains tested per type of agar. The number of 137 grown colonies was counted on each plate after 48 hours of incubation for the calculation of the yield in 138 colonies forming unit (CFU) per mL. When the number of colonies exceeded 100, it would be scored as 139 “> 100”. 140 141 Detection of CRPA from spiked faeces cultured in enrichment broths with subculturing on selective 142 agar plates 143 Spiking experiments were performed to further evaluate the best performing enrichment broths in 144 combination with the best performing selective agar plates (Figure 1). Faecal samples without 145 carbapenem-resistant bacteria were used for these experiments. Faecal solutions were prepared by 146 suspending 5 grams of patients’ faeces into 50 mL of sterile distilled water. The spiked samples were 147 made by adding 100 µL of the CRPA strain suspensions to 900 µL of the faecal suspension. In addition to 148 the seven well-characterized strains of CRPA mentioned previously, suspensions of carbapenem-149 susceptible P. aeruginosa (CSPA) ATCC 27853 and Aeromonas caviae ATCC 15468 were also used. The 150 latter was chosen as this microorganism is often present in water samples, and our detection method 151 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 8 was developed in a One Health project, also focused on water. As a negative control, 100 µL of 152 physiological salt (0.85%) was added to 900 µL of the faecal suspension. 153 Following the preparatory steps, 100 µL of the spiked samples and negative control were added to 154 the selected enrichment broths and incubated for 24 hours at 35±1°C. The next day, 100 µL of the broth 155 was sub-cultured onto the selected selective agar plates and spread evenly. The plates were then 156 incubated for 18, 24, and 48 hours at 35±1°C. Colonies were identified using MALDI-TOF MS, followed by 157 testing the susceptibility to carbapenems using the disc diffusion test for P. aeruginosa according to 158 EUCAST. The growth of CRPA and other microorganisms on the plate was recorded. The experiment was 159 performed three times with different faecal samples. 160 161 162 Figure 1 Overview of enrichment broths and agar plates tested, and spiking experiments performed. 163 PIA Pseudomonas isolation agar, TSB tryptic soy broth. 164 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 9

165 Comparison of enrichment broths 166 TSB-vancomycin and TSB-vancomycin supplemented with 2 mg/L imipenem both had 100% 167 sensitivity with the dilutions of 10-5 and 10-6 (Table 2). Even though the sensitivity of TSB-vancomycin 168 and TSB-vancomycin supplemented with 2 mg/L imipenem decreased to 85.7% and 71.4%, respectively, 169 with the dilution of 10-7, each strain could be recovered in at least one of the experiments. For TSB-170 vancomycin supplemented with 4 mg/L imipenem, the sensitivity already decreased to 85.7% in the 10-6 171 dilution, whereas for the TSB-vancomcyin supplemented with 6 mg/L ceftazidime the sensitivity was 172 only 76.2% with the 10-5 dilution. Overall, the sensitivities of TSB-vancomycin and TSB-vancomycin 173 supplemented with 2 mg/L of imipenem were highest with the different dilutions and were selected for 174 further testing. 175 Table 2 Evaluation of four different enrichment broths with seven well-characterized carbapenem-176 resistant Pseudomonas aeruginosa strains, each tested in triplo. 177 Dilutions Sensitivity TSB-vancomycin TSB-vancomycin + 2 mg/L imipenem TSB-vancomycin + 4 mg/L imipenem TSB-vancomycin + 6 mg/L ceftazidime 10-5 21/21 - 100% 7/7 strains 21/21 - 100% 7/7 strains 21/21 - 100% 7/7 strains 16/21 - 76.2% 6/7 strains 10-6 21/21 - 100% 7/7 strains 21/21 -100% 7/7 strains 18/21 - 85.7% 7/7 strains 13/21 - 57.1% 5/7 strains 10-7 18/21 - 85.7% 7/7 strains 15/21 - 71.4% 7/7 strains 5/21 - 23.8% 4/7 strains 7/21 - 33.3% 4/7 strains 10-8 0/21 - 0% 0/7 strains 3/21 - 14.3% 3/7 strains 3/21 - 14.3% 3/7 strains 1/21 - 4.8% 1/7 strains TSB-vancomycin tryptic soy broth supplemented with 2 mg/L of vancomycin. 178 179 Comparison of selective agar plates 180 All strains were able to grow on all selective agar plates after 24 hours of incubation in experiment 181 with the dilution of 10-5 (1.5 × 102 CFU/mL), with no additional colonies observed on all the agar plates 182 after 48 hours. However, P. aeruginosa colonies were better recognizable after 42 hours (Figure 2). 183 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 10 184 Figure 2 Growth of carbapenem-resistant Pseudomonas aeruginosa on different agar plates after 42 185 hours of incubation. 186 The agar plates shown in Figure 2 are blood agar (1), ChromID (2), Pseudosel (3), Cetrimide Oxoid (4), 187 Cetrimide bioMérieux (5), PIA Becton Dickinson (6), PIA Sigma-Aldrich (7), M-PA-C agar (8), and 188 Phenanthroline agar (9). The images show the growth of strain 2, 10-6 dilution, after 42 hours of 189 incubation. 190 Figure 2 shows the colony morphologies on different selective agar plates with blood agar as the 191 control. P. aeruginosa colonies were yellow-brown on Cetrimide Oxoid, Cetrimide bioMérieux, PIA 192 Becton Dickinson, PIA Sigma-Aldrich, and Phenantroline agar plates. On ChromID agar plates, colonies 193 had purplish-pink pigmentation with dark blue centers, while M-PA-C agar plates showed pinkish-194 pigmentation with dark centers. The colonies were bright greenish-yellow on the Pseudosel agar plates. 195 1 2 3 4 5 6 7 8 9 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 11 Table 3 Number of strains growing on the plates. 196 Dilution ChromID Pseudosel Cetrimide Oxoid Cetrimide bioMérieux PIA BD PIA SA M-PA-C Phenan- throline Blood agar 18 hours 10-5 7 of 7 (100%) 6 of 7 (86%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 10-6 6 of 7 (86%) 6 of 7 – (86%) 6 of 7 (86%) 5 of 7 (71%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 10-7 3 of 7 (43%) 1 of 7 – (14%) 2 of 7 (29%) No growth 1 of 7 – (14%) 3 of 7 (43%) 1 of 7 (14%) 1 of 7 (14%) 2 of 7 (29%) 10-8 No growth No growth No growth No growth No growth No growth 1 of 7 (14%) No growth 1 of 7 (14%) 24 hours 10-5 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 10-6 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 10-7 4 of 7 (57%) 2 of 7 (29%) 4 of 7 (57%) 2 of 7 (29%) 2 of 7 – (29%) 5 of 7 (71%) 3 of 7 (43%) 1 of 7 (14%) 2 of 7 (29%) 10-8 1 of 7 (14%) No growth 2 of 7 (29%) No growth 1 of 7 – (14%) 2 of 7 (29%) No growth No growth 1 of 7 (14%) 42 hours 10-5 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 10-6 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 10-7 4 of 7 (57%) 2 of 7 (29%) 4 of 7 (57%) 2 of 7 (29%) 2 of 7 – (29%) 5 of 7 (71%) 3 of 7 (43%) 1 of 7 (14%) 2 of 7 (29%) 10-8 1 of 7 (14%) No growth 2 of 7 (29%) No growth 1 of 7 – (14%) 2 of 7 (29%) No growth No growth 1 of 7 (14%) 48 hours 10-5 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 10-6 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 10-7 4 of 7 (57%) 2 of 7 (29%) 4 of 7 (57%) 2 of 7 (29%) 2 of 7 – (29%) 5 of 7 (71%) 3 of 7 (43%) 1 of 7 (14%) 2 of 7 (29%) 10-8 1 of 7 (14%) No growth 2 of 7 (29%) No growth 1 of 7 – (14%) 2 of 7 (29%) No growth No growth 1 of 7 (14%) 72 hours 10-5 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 10-6 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 7 of 7 (100%) 7 of 7 (100%) 5 of 7 (71%) 7 of 7 (100%) 10-7 4 of 7 (57%) 2 of 7 (29%) 4 of 7 (57%) 2 of 7 (29%) 2 of 7 – (29%) 5 of 7 (71%) 3 of 7 (43%) 1 of 7 (14%) 2 of 7 (29%) 10-8 1 of 7 (14%) No growth 2 of 7 (29%) No growth 1 of 7 – (14%) 2 of 7 (29%) No growth No growth 1 of 7 (14%) BD Becton Dickinson, PIA Pseudomonas Isolation Agar, SA Sigma-Aldrich. 197 The darkest shade of green shows the most sensitive plate, the lighter the shade the lower the 198 sensitivity. 199 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 12 Table 3 shows the sensitivity of the selective agar plates and the blood agar plates. At 18 hours of 200 incubation, PIA Sigma Aldrich had the highest sensitivity with the dilutions up to 10-7. With that dilution, 201 PIA Sigma Aldrich was more sensitive compared to the blood agar plate (i.e., the current gold standard). 202 After 18 hours of incubation, no additional growth was observed on the blood agar and Phenanthroline 203 agar plates, while the sensitivity increased for the other selective agar plates. After 24 hours of 204 incubation, there was only one additional colony growth from each cetrimide bioMérieux (observed 205 after 42 hours of incubation), PIA Becton Dickinson and PIA Sigma-Aldrich plate (both were observed 206 after 48 hours of incubation). No additional growth was seen for incubation periods longer than 48 207 hours. Compared to the blood agar plate, ChromID, Cetrimide Oxoid, PIA Becton Dickinson, PIA Sigma-208 Aldrich, and M-PA-C all had higher sensitivities across the different incubation times and/or dilutions. 209 Overall, PIA Sigma Aldrich had the highest sensitivity, followed by Cetrimide Oxoid and ChromID. 210 Pseudosel, Cetrimide bioMérieux, and Phenanthroline agar plates did not perform well and no CRPA 211 from the 10-8 dilution grew. All three experiments showed consistent results. 212 213 Table 4 Yields of growth on plates after 48 hours of incubation. 214 Strain Yield (x 10-6 CFU/mL) Blood Agar ChromID Pseudosel Cetrimide Oxoid Cetrimide bioMérieux PIA BD PIA SA M-PA-C Phenan- throline 1 1.10 1.10 1.23 1.60 1.07 1.40 1.17 1.60 1.60 2 1.77 0.87 0.87 0.95 1.28 1.97 1.37 1.93 0.50 3 1.23 1.07 1.57 0.93 1.40 1.67 1.27 1.33 0.78 4 0.60 0.74 0.06 0.63 0.30 0.98 0.94 0.92 0.04 5 1.50 1.80 1.95 2.10 1.60 1.80 2.15 2.10 0.29 6 2.10 1.00 0.91 1.00 1.90 1.50 1.75 2.30 0.61 7 1.38 0.65 0.40 0.65 0.49 0.93 1.10 0.55 0.01 Average 1.38 1.03 1.00 1.12 1.15 1.46 1.39 1.53 0.55 BD Becton Dickinson, PIA Pseudomonas Isolation Agar, SA Sigma Aldrich. 215 Green = higher yield than blood agar (control) 216 217 Table 4 shows the number of CFU/mL for each strain on each agar plate. For some strains, some 218 agar plates failed to yield a higher number of colonies than the standard blood agar. For instance, 219 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 13 phenanthroline agar only yielded a higher number of colonies of the strain 1. None of the selective agar 220 plates yielded a higher number of colonies of strain 7 compared to blood agar. On average, the M-PA-C 221 agar had the highest yield (1.53), while the Phenanthroline agar had the lowest yield (0.55). There were 222 three agar plates with higher yields than blood agar, M-PA-C, PIA Becton Dickinson and PIA Sigma-223 Aldrich. Based on the combination of sensitivity and yield of each plate, those three were selected for 224 the spiking experiments. 225 226 Growth of CRPA from human faecal samples spiked with CRPA (spiking experiment) 227 For these experiments, the two most sensitive enrichments broths ( i.e., TSB-vancomycin and TSB -228 vancomycin with 2 mg/L imipenem ) and the most sensitive selective agar plates ( i.e., PIA Becton 229 Dickinson, PIA Sigma-Aldrich, and M -PA-C) were combined (Table 5). All selective agar plates used in 230 combination with TSB -vancomycin with 2 mg/L imipenem had the same sensitivity in all samples. The 231 combination of TSB-vancomycin and M-PA-C had the highest sensitivity in detecting CRPA in Sample 1 and 232 3. No CSPA was detected when TSB-vancomycin with 2 mg/L imipenem was used. As expected, CSPA was 233 found when using a broth without antibiotics, which hampered growth of CRPA. In all samples and all 234 experiments, there was no Pseudomonas spp. other than P. aeruginosa found. The agar plate with the 235 least amount of other growth was M-PA-C. All six methods allowed yeasts, such as Candida albicans and 236 Candida tropicalis, to grow. 237 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 14 Table 5 Evaluation of the combination of different enrichment broths and selective agar plates for the detection of carbapenem-resistant 238 Pseudomonas aeruginosa 239 Number of CRPA strains marked positive TSB-Vancomycin TSB-Vancomycin + Imipenem 2 mg/L PIA BD PIA SA M-PA-C PIA BD PIA SA M-PA-C Sample 1 Negative control Escherichia coli Enterococcus faecium Escherichia coli Escherichia coli Unidentified No growth Escherichia coli CSPA ATCC 27853 10-5 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-6 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-7 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth CSPA CSPA CSPA None detected None detected None detected Escherichia coli Escherichia coli Escherichia coli Klebsiella oxytoca Aeromonas caviae ATCC 15468 10-5 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-6 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-7 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth Aeromonas caviae Enterococcus casseliflavus Escherichia coli Clostridium tertium Unidentified None detected Escherichia coli Kocuria rhizophila Klebsiella pneumoniae CRPA (Strain 1-7) 10-5 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 10-6 5 of 7 – 71.4% 6 of 7 – 85.7% 5 of 7 – 71.4% 3 of 7 – 42.9% 3 of 7 – 42.9% 3 of 7 – 42.9% 10-7 No CRPA detected No CRPA detected 2 of 7 – 28.6% 1 of 7 – 14.3% 1 of 7 – 14.3% 1 of 7 – 14.3% 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth Enterococcus casseliflavus Enterococcus casseliflavus Escherichia coli Clostridium tertium Enterococcus casseliflavus Micrococcus luteus Escherichia coli Escherichia coli Enterococcus casseliflavus Enterococcus spp. Klebsiella pneumoniae Klebsiella pneumoniae Enterococcus spp. Lactobacillus gasseri .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 15 Micrococcus luteus Klebsiella oxytoca Micrococcus luteus Micrococcus luteus Paenibacillus spp. Sample 2 Negative control Escherichia coli Enterococcus faecium Escherichia coli Escherichia coli Unidentified No growth Escherichia coli CSPA CSPA ATCC 27853 10-5 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-6 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-7 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth CSPA CSPA CSPA Candida tropicalis Candida albicans Candida tropicalis Enterococcus faecalis Candida tropicalis Escherichia coli Enterococcus faecalis Aeromonas caviae ATCC 15468 10-5 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-6 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-7 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth CSPA CSPA CSPA CSPA Enterococcus raffinosus CSPA Enterococcus raffinosus Enterococcus faecalis Escherichia coli CRPA (Strain 1-7) 10-5 No CRPA detected No CRPA detected No CRPA detected 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 10-6 No CRPA detected No CRPA detected No CRPA detected 5 of 7 – 71.4% 5 of 7 – 71.4% 5 of 7 – 71.4% 10-7 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth CSPA CSPA CSPA Candida albicans Candida albicans Candida albicans Enterococcus faecalis Enterococcus faecalis Candida tropicalis Candida tropicalis Candida tropicalis Candida tropicalis Enterococcus faecalis Enterococcus faecalis Enterococcus faecalis Enterococcus raffinosus Escherichia coli Sample 3 Negative control Citrobacter freundii Citrobacter freundii Candida albicans Candida albicans Candida albicans Candida albicans Lactobacillus fermentum Lactobacillus paracasei .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 16 CSPA ATCC 27853 10-5 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-6 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-7 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth Citrobacter freundii Citrobacter freundii Candida albicans Candida albicans Candida albicans Candida albicans CSPA CSPA Lactobacillus paracasei Lactobacillus plantarum Escherichia coli Aeromonas caviae ATCC 15468 10-5 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-6 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-7 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth Aeromonas caviae Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Citrobacter freundii Citrobacter freundii Lactobacillus murinus Haemophilus parainfluenzae Lactobacillus paracasei CRPA (Strain 1-7) 10-5 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 7 of 7 – 100% 10-6 2 of 7 – 28.6% 6 of 7 – 85.7% 5 of 7 – 71.4% 2 of 7 – 28.6% 2 of 7 – 28.6% 2 of 7 – 28.6% 10-7 No CRPA detected No CRPA detected 1 of 7 – 14.3% 1 of 7 – 14.3% 1 of 7 – 14.3% 1 of 7 – 14.3% 10-8 No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected No CRPA detected Other growth Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Citrobacter freundii Citrobacter freundii Escherichia coli Citrobacter freundii Mold Escherichia coli Escherichia coli Lactobacillus murinus Escherichia coli Haemophilus parainfluenzae Haemophilus parainfluenzae Lactobacillus paracasei Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus murinus Lactobacillus murinus Lactobacillus plantarum Lactobacillus plantarum BD Becton Dickinson, CSPA carbapenem-susceptible Pseudomonas aeruginosa, PIA Pseudomonas Isolation Agar, SA Sigma Aldrich. 240 The darkest shade of green shows the most sensitive combination, the lighter the shade the lower the sensitivity. 241 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 17

242 This study shows that the best method for the detection of CRPA is by inoculating the sample in TSB 243 -vancomycin supplemented with 2 mg/L imipenem, continued by subculturing the broth onto the M-PA-244 C agar plate. Imipenem supplementation in the enrichment broth was efficiently eliminating CSPA, even 245 when CSPA was intentionally added. When the sample naturally contained CSPA, it masked the CRPA 246 spiked into the sample. As a result, the methods without imipenem supplementation failed to grow the 247 CRPA. Despite having a lower sensitivity in higher dilutions, the combination of TSB-vancomycin with 248 imipenem and M-PA-C agar plate resulted in the least amount of growth of other microorganisms. 249 Use of a selective enrichment broth has proven to be useful for the detection of carriage of various 250 multidrug-resistant microorganisms, such methicillin-resistant Staphylococcus aureus and vancomycin-251 resistant Enterococcus faecium (22, 23). For CRPA, there is only one report evaluating the use of an 252 enrichment broth supplemented with antibiotics (i.e., meropenem) (24). However, as mentioned in the 253

section, imipenem was chosen as the carbapenem of choice, because a study reported its 254 stability compared to meropenem (19). 255 Selective agar plates are useful as they promote growth of a specific microorganism, in our case P. 256 aeruginosa, while inhibiting other species. Thus, identification of CRPA would be feasible. Moreover, the 257 increased pigment production resulting from culturing P. aeruginosa on selective agar plates containing 258 magnesium chloride and potassium sulfate makes the identification easier (Supplementary Table 1). 259 Among eight different selective agar plates tested, M-PA-C and PIA Sigma-Aldrich showed high 260 sensitivities (Table 3) and yields (1.53 and 1.39 × 10-6 CFU/mL, respectively). One of the differences 261 between these two media is the addition of nalidixic acid to inhibit other Gram-negative bacteria and 262 kanamycin as selective agent to inhibit the growth of Gram-positive bacteria in the M-PA-C agar. A 263 previous study showed that medium containing nalidixic acid and kanamycin has a higher sensitivity in 264 detecting P. aeruginosa compared to other selective media (21). 265 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 18 In the spiking experiment using TSB-vancomycin, the growth of E. coli was found in sample 1 and 266 sample 3 on all agar plates tested. E. coli is generally susceptible to nalidixic acid (selective component 267 of M-PA-C) and some strains of E. coli are reported to be resistant against triclosan (selective component 268 of PIA) (25, 26). In the manufacturers’ guide of all agar plates tested, E. coli ATCC 25922 is reported as 269 the quality control strain and should result in partial to complete inhibition. When imipenem was added 270 to the enrichment broth, the growth of E. coli was inhibited and only detected in sample 2 (on M-PA-C) 271 and sample 3 (on PIA Becton Dickinson plate). Yeast could not be eliminated by the six methods tested 272 and could potentially interfere with CRPA growth. Colonies, however, can be easily recognized as yeasts. 273 Spiking experiments simulate how the method performs when applied to human samples. 274 Numerous bacteria in the normal flora may obscure the low number of CRPA in nonselective culture 275 methods. When adding either CSPA or A. caviae, it was shown that these were suppressed by the 276

with TSB-vancomycin supplemented with imipenem 2 mg/L. 277 The broth used in this study can be made by adding antibiotic discs to the broth, generally used in 278 laboratories around the world. The selective agar with the highest sensitivity in this study can be 279 shipped and stored easily because it is sold as powder. Thus, the proposed method is feasible for 280 laboratories with limited resources or in remote areas. Furthermore, because of its high sensitivity, this 281

can be used in surveillance or screening of CRPA in healthy people as well. 282 This study has some limitations. First, Pseudomonas spp. other than P. aeruginosa were not 283 included in the spiking experiment. The selective agar plates used in this study are selective for P. 284 aeruginosa and supposedly able to suppress the growth of those species or at least make the other 285 colonies colorless. Second, only seven strains have been used in this study, but they all are important 286 high-risk clones of CRPA. Third, our study was focused on screening with rectal or faecal samples and we 287 did not include other body sites which can be useful for screening, such as throat. Finally, faecal samples 288 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 19 used in the spiking experiment were from Dutch persons and their microbiota might be different from 289 persons in other countries. 290 291

292 In this study, a highly-sensitive method to detect carriage of CRPA was developed using a stepwise 293 approach. The best method for detecting CRPA based on sensitivity and selectivity was the combination 294 of TSB-vancomycin with 2 mg/L imipenem as an enrichment broth for overnight incubation, followed by 295 subculturing the broth onto an M-PA-C agar plate. Careful colony selection followed by identification 296 and susceptibility testing is needed after a positive result to confirm the screening results. Real 297 implementation of the screening of CRPA in humans, where the number of CRPA might be limited and 298 affected by a variety of normal flora, is needed to verify the clinical use and the practicality. 299 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 20 CRediT AUTHORSHIP CONTRIBUTION STATEMENT 300 S.N.S.: formal analysis, visualization, writing – original draft, writing – review & editing 301 A.V.: project administration, writing – review & editing 302 N.K: investigation, resources, validation, writing – review & editing 303 A.R.: investigation, resources, validation, writing – review & editing 304 H.S.: supervision, writing – review & editing 305 Y.R.S: writing – review & editing 306 M.C.V.: supervision, writing – review & editing 307 A.K.: funding acquisition, supervision, writing – review & editing 308 J.A.S.: conceptualization, formal analysis, funding acquisition, methodology, supervision, validation, 309 writing – review & editing 310 311 DECLARATION OF COMPETING INTERESTS 312 The authors declare that they have no known competing financial interests or personal relationships 313 that could have appeared to influence the work reported in this paper. 314 315

316 The authors are grateful to all members of the SAMPAN Consortium for their input: Anniek de Jong 317 (Deltares, Delft, the Netherlands) and Roger C. Lévesque (U. Laval Integrative Systems Biology Institute, 318 Québec, Canada). Also, the authors would like to acknowledge the National Institute for Infectious 319 Diseases “L. Spallanzani” IRCCS, Rome, Italy, for their contribution in the study design (Enrico Girardi). 320 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 21 FUNDING 321 This work was part of the SAMPAN project (A Smart Surveillance Strategy for Carbapenem-resistant 322 Pseudomonas aeruginosa), which was financially supported by JPIAMR 9th call, Dutch ZonMw (grant no. 323 549009005). S.N.S. was supported by an Erasmus+ scholarship (funding ID: 587538). S.N.S., Y.R.S., and 324 A.K. were supported by International Development Research Centre (grant no. 109283-001). 325 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 27, 2024. ; https://doi.org/10.1101/2024.08.27.609846doi: bioRxiv preprint 22

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