Peripheral Parenteral Nutrition Solutions and Bed Bath Towels as Risk Factors for Nosocomial Peripheral Venous Catheter-related Bloodstream Infection by Bacillus cereus

In Japan, China, and Singapore, several studies have reported increased incidences of peripheral venous catheter-related bloodstream infection by Bacillus cereus during the summer. Therefore, we hypothesized that bed bathing with a B. cereus-contaminated “clean” towels increases B. cereus contact with the catheter and increases the odds of contaminating the peripheral parenteral nutrition (PPN). We found that 1) professionally laundered “clean” towels used in hospitals have B. cereus (3.3×104 colony forming units (CFUs) / 25cm2), 2) B. cereus is transferable onto the forearms of volunteers by wiping with the towels (n=9), and 3) B. cereus remain detectable (80∼660 CFUs /50cm2) on the forearms of volunteers even with subsequent efforts of disinfection using alcohol wipes. We further confirmed that B. cereus grow robustly (102 CFUs /mL to more than 106 CFUs /mL) within 24hours at 30°C in PPN. Altogether we find that bed bathing with a towel contaminated with B. cereus leads to spore attachments to the skin, and that B. cereus can proliferate at an accelerated rate at 30°C compared to 20°C in PPN. We therefore highly recommend ensuring the use of sterile bed bath towels prior to PPN administration with catheter in patients requiring bed bathing.


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However, the reason why B. cereus-related septicemia frequently develops in the latter, the summer season, remains to be clarified. In this study, we examined the etiology of a high incidence of B. cereus-induced PVC-BSI in the summer.

Microorganisms employed
The following strains were used: B. cereus ATCC 11778, a total of 28

Viability of microorganisms in infusion solutions
All microbes used in the experiment were cultured on trypticase soy agar (TSA; Eiken Chemical Co., Tochigi, Japan) for 1-2 days at 35°C, scraped into sterile phosphate-buffered saline (PBS), and centrifuged three times at 3,000 rpm for 10 min to remove the growth medium. Resuspension was carried out in PBS, yielding a concentration of approximately 10 4 -10 5 colony forming units (CFUs)/mL. Next, 0.05 mL of the resuspension was added to 4.95 mL of each infusion solution. The test solutions were incubated at 20 and 30°C, and plate counts were performed at 6, 24, 48 h. Each sample was diluted 10-, 10 2 -, 10 3 -, 10 4 -, 10 5 -, and 10 6 -fold in normal saline. Pipettes were used to transfer 0.25 mL of undiluted or diluted samples to TSA. The plates were streaked with a glass 'hockey stick' and incubated at 35°C for 1-2 days for measurement of the number of viable microorganisms. Each experiment was repeated 3 times and the mean of the 3 repeats was calculated.

Contaminated towels with B. cereus
We had initially observed that hospital bed bath towels shipped from the laundry service factory yield B. cereus. The bed bath towels were prepared by the laundry service, washing in high temperature at 80 °C for 10 minutes, per regulatory guidelines in Japan. Approximately 1.3 × 10 3 CFUs / cm 2 of B. cereus was found on the towels.

Transfer of B. cereus from a contaminated towel to the skin, and disinfection of B. cereus on the skin by alcohol
This study was conducted with the participation of volunteer pharmaceutical students and staff from the Sanyo-Onoda City University Ethics Review Committee (Title: Attachment of B. cereus to the skin related to bed bathing with B. cereus-contaminated towels, Approval date: September, 2022, Examination certificate management number: 22001).
In 12 subjects (volunteers), we initially screened for presence or absence of B. cereus on subject skin by wiping bilateral forearms (5 cm × 10 cm) with a piece of wet sterile gauze (5 cm × 5 cm). This gauze was placed in a bottle containing 20 mL of sterile physiological saline, and ultrasonically treated at 37kHz for 5 min. Subsequently, the solution in the bottles was diluted 10-fold in normal saline. Pipettes were used to transfer 1 mL (0.25 mL × 4) of undiluted or diluted samples to PBCW agar (Eiken Chemical Co., Ltd.) containing egg yolk. The plates were streaked with a glass 'hockey stick' and incubated at 35°C for 24 h. Additionally, the residual solution was passed through 0.22 µm membrane filters, 5 cm in diameter (Thermo Scientific Nalgene Analytical Filter Unit 0.2 µm, Thermo Scientific, Wilmington, NC, USA), and the filters were placed on PBCW agar containing egg yolk. After incubation at 35°C for 24 h, the colonies of B. cereus were counted [29].
Next, the following experiment was conducted in the 9 volunteers that we found B. cereus to be absent on the forearms. Two pieces (5 cm × 5 cm) of bed bath towel that were found to be contaminated with 1.3 × 10 3 CFUs/cm 2 of B. cereus were each dampened with 2mL of sterile purified water. Using each piece, each subject's bilateral forearms (5 cm × 10 cm) were wiped. After naturally air drying, the unilateral forearm was wiped with sterile-water-drenched gauze, and the amount of B. cereus in this gauze was investigated to calculate the forearm B. cereus [30].
Additionally, the other forearm was wiped twice with a medical-grade absorbent cotton (4 cm × 8 cm) containing 1.6 mL of ethanol for disinfection (76.9-81.4 vol%), and wiped with a piece of sterile-water-drenched gauze after 1 min. Subsequently, the amount of B. cereus in this gauze was investigated to calculate the forearm CFUs of B.
cereus after alcohol disinfection. The amount of B. cereus in the gauze used for wiping was calculated as described above. In 9 volunteers, this test was conducted twice. The forearm CFUs of B. cereus before and after alcohol disinfection was tested using Wilcoxon's signed-rank test.

Results
The viability of B. cereus in various types of infusion fluid are shown in Figure 1. At 30°C, B. cereus promptly proliferated in all 3 types of PPN we tested. Furthermore, B. cereus also proliferated in soybean oil or albumin, but not in acetated Ringer's solution, normal saline, 5% glucose, or total parenteral nutrition. On the other hand, the growth rate at 20°C was slower than at 30°C. However, B. cereus proliferated robustly by the 48-hour timepoint in PPN, soybean oil, and albumin, as demonstrated at 30°C. The viability of 28 clinical isolates of B. cereus in PPN (Bfluid ® ) are shown in Figure 2. These isolates promptly proliferated in PPN, as shown for a standard strain of B. cereus in Figure 1. After 24 h, these isolates had more rapidly proliferated at 30°C, but the CFU of bacteria after 48 h was similar between 30°C and 20°C.
The viability of various microorganisms in PPN (Bfluid ® ) at 30°C and 20°C are shown in Figure 3. Of the 5 strains that we investigated, 4 (E. coli, K. pneumoniae, P. aeruginosa, S. marcescens), excluding C. albicans, promptly proliferated in PPN at 30°C and 20°C, as demonstrated for B. cereus.

Discussion
In Japan, double-bag-type PPN products are routinely used. It was shown that a standard strain of B. cereus and its clinical isolates rapidly proliferated in 3 double-bag-type PPN products commercially available in Japan. The growth rate with PPN was equally robust as with fat emulsion or blood preparations (albumin). In this study we demonstrated that B. cereus proliferates in PPN, in addition to other organisms such as E. coli, S. marcescens, K. pneumoniae, and P. aeruginosa, reported by Omotani et al. [31]. In the CDC Guideline for the Prevention of Intravascular Catheter-Related Infections, it is prescribed that tubing used to administer blood, blood products, or lipid emulsion should be replaced within 24 h after the initiation of infusion [32]. Such a regulation may be necessary for PPN products (excluding one-bag-type acidic-pH products).
We had initially observed that hospital bed bath towels shipped from the laundry service factory yield B. cereus. The bed bath towels were prepared by washing in high temperature at 80°C for 10 min, per regulatory guidelines in Japan. We determined that the washed "clean" bed bath towels we tested harbored approximately 1.3×10 3 CFUs/cm 2 of B. cereus, despite undergoing recommended cleaning protocol set by industry-wide practice. Using these "clean" towels to wipe the volunteer forearms, we determined that 6.4 CFUs (median)/cm 2 of B. cereus spores remained on the left and 4.8 CFUs (median)/cm 2 on the right skin after wiping with contaminated bed bath towels, despite disinfecting the skins with ethanol wipes afterwards. Our calculation of B. cereus contamination via catheter placement, using the median CFUs /cm 2 values, is the following: If a venous indwelling needle (22 G; catheter's cross-sectional area, 0.64 mm 2 ) is inserted to the skin, since the cross-sectional area of the catheter is 0.64 mm 2 , B. cereus spores may contaminate the indwelling needle at a probability of 0.41% on the left arm and 0.31% on the right arm. If B. cereus contaminates the needle or the site of catheter placement, bloodstream infection is a possible outcome due to rapid proliferation in PPN.
The amount of B. cereus found in the bed bath towel used in this experiment was approximately 10 3 CFUs /cm 2 . However, in Japan with a climate of high temperature and high humidity, it is not rare that towels after washing in a cleaning factory may still harbor a higher concentration (10 4 CFUs /cm 2 ) of B. cereus. If a towel contaminated with approximately 10 4 CFUs /cm 2 of B. cereus was used for a bed bath, the probability at which B. cereus may access the venous indwelling needle will potentially be higher at 3.1 -4.1% even after wiping the skin with ethanol. Ethanol is ineffective for killing B. cereus spores, and we show here that B. cereus has the potential to proliferate in the infusion fluids.
The detection rate of B. cereus in blood culture-positive samples is 1.2% according to the information published from the Japan Nosocomial Infections Surveillance (JANIS) in 2020. The rates in A and B Hospitals (736 and 904 beds, respectively) were 1.7 and 22.2%, respectively [28]. It is highly probable that bed bath towels contaminated with B. cereus may have contributed to the high outbreak rates of B. cereus in B Hospitals measured in the blood-culture-positive samples. Patients positive for B. cereus on blood culture were mostly patients requiring bed baths. B Hospital's bed bath towels used for bed bathing were from a cleaning factory. Several studies indicated that bed bath towel contamination with B. cereus was frequent in the summer of Japan, China, and Singapore [3][4][5]33]. This may be because towels are often contaminated with B. cereus in the summer of Japan, China, and Singapore with a climate of high temperature and high humidity. The bed bath towels used in A Hospitals were independently confirmed by us to have absence of contamination with B. cereus.
B. cereus is regarded as lowly-pathogenic environmental bacteria [34]. However, if infusion fluids such as PPN, is contaminated with B. cereus, this type of bacteria may proliferate, and large numbers of bacteria may invade the body, leading to a serious outcome. We encountered a patient with sepsis in whom 1.0×10 7 CFUs/mL of B. cereus was detected in the route of administration during PPN administration [17]. Furthermore, several studies indicated that B. cereus sepsis was very serious [6,7,12,14,15,16,19,20,35], that there was no treatment response despite the administration of effective antimicrobial drugs [9,21], and that catheter removal was required [8,36], suggesting intra-route infusion fluid contamination with bacteria. In many hospitals, infusion fluid contamination with microorganisms during administration is not investigated and is often overlooked. However, physicians must recognize that infusion fluid may be contaminated with microorganisms during use.
We previously reported that measurement of the adenosine triphosphate (ATP) level was useful as a simple method to evaluate infusion fluid contamination with microorganisms [17,37]. Since infusion fluid contamination can be estimated in a few minutes by measuring the ATP level, infusion fluid that is being administered should be checked using a simple detection method with ATP in patients in whom sepsis is suspected based on findings, such as a high procalcitonin level.
The inadvertent use of contaminated "clean" towels must be avoided, and to ensure quality control, laundry services for hospitals should be required to pass periodic pathogen screening to be given permission to supply and stock sterile towels. In addition, disposable wipes in turn may be particularly useful in the summer seasons. The implementation of standard guidelines that indicate use of sterile bed bath towels or disposable wipes for patients that require PPN would be beneficial for avoiding preventable infections.

Conclusion
The use of B. cereus-contaminated bed bath towels and administration of double-bag-type PPN products may be involved in higher incidence of B. cereus sepsis especially during the summer months in countries such as Japan, China, and Singapore. We demonstrate the ability of B. cereus to proliferate rapidly in PPN products and that higher temperatures lead to faster proliferation rate. If double-bag-type PPN products are contaminated with B. cereus, it is possible that B. cereus may proliferate rapidly in the host via catheter access point. Therefore, the use of sterile bed bath towels must be strongly considered for patients that require PPN.