Impact of environmental cleaning on the colonization and infection rates of multidrug-resistant Acinetobacter baumannii in patients within the intensive care unit in a tertiary hospital

doi:10.21203/rs.2.19645/v1

Objective: To continuously evaluate the effect of environmental cleaning on the colonization and infection rates of multidrug-resistant Acinetobacter baumannii (MDR-AB) in the patients within a intensive care unit (ICU).

Methods: Environmental cleaning on the high-touch clinical surfaces (HTCS) within a comprehensive ICU was evaluated through monitoring fluorescent marks. In the meanwhile, samples from the HTCS and inpatient were collected and sent for bacterial culture and identification. The drug susceptibility testing was further implemented to monitor the prevalence of MDR-AB. The genetic relatedness of MDR-AB collected either from the HTCS or inpatient was analyzed by pulsed field gel electrophoresis (PFGE) when an outbreak was doubted.

Results: From the beginning to the end of 2013, the clearance rate of fluorescence marks on the environmental surfaces within ICUs significantly increased from 21.9% to 85.7%, and accordingly the colonization and infection rates of MDR-AB decreased from 16.5‰ to 6.6‰ and 7.4‰ to 2.8‰, respectively. However, during the year 2014, because of frequent change and movement of cleaning workers, the clearance rate of fluorescence marks decreased below 50%, and the overall colonization and infection rates of MDR-AB correspondingly increased from 9.1‰ to 11.1‰, 1.5‰ to 3.9‰, respectively. PFGE displayed a genetic relatedness between the MDR-AB strains analyzed, indicating a dissemination of MDR-AB during the surveillance period.

Conclusion: For MDR-AB, both the colonization rates and the infection rates of the patients within ICUs can be directly influenced by environmental cleaning, and the bad environmental cleaning may result in MDR-AB outbreak. The invisible fluorescence labelling is an effective and convenient method to continuously monitor environmental cleaning within hospitals.

General Microbiology

Infectious Diseases

healthcare-associated infection

multidrug resistance

Acinetobacter baumannii

fluorescence labeling

environmental cleaning

Healthcare-associated infection (HAI) is a global problem for patients, especially those inpatient with immunocompromised or critically ill diseases, causing extended hospital stays, high costs, and high mortality (1). Epidemiological studies showed that HAI is closely associated with microbial pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) (2, 3), vancomycin-resistant Enterococci (VRE) (3), and multidrug-resistant Gram-negative bacilli (4), which could be spread by the hospital environment surfaces (5). In USA and Europe, MRSA and VRE are the main pathogens associated with HAI within ICUs (3, 6, 7). In contrast, in China, the prevalence of MDR-AB greatly exceeds both MRSA and VRE (8, 9), and the frequent expansion of MDR-AB poses tough challenges to control HAIs, especially those occurred in ICUs (10) (11). A. baumannii possesses the super survivability on kinds of healthcare equipment surfaces, from 5 days to more than 5 months (12), which surely increases the chance of transmission of MDR-AB. In addition, such strain is difficult to eliminate once it acquires resistance to the conventional detergents and alcohol disinfectants (13). The worrying condition is, the current arsenal to target MDR-AB is almost exhausted (14). Therefore, management of A. baumannii clusters in hospitals is the very important part of eradication of multidrug-resistant infections. The targeted infection control measures of MDR-AB including intensive cleaning and subsequent measurement of cleanliness are imperative to prevent the nosocomial acquisition and further dissemination.

To date, it’s known that the patient care items and environmental surfaces frequently touched (high-touch clinical surfaces, HTCS) within hospital wards are blamed for pathogen transmission (15), and the cleaning and disinfection of HTCS are pivotal for prevention of outbreak of MDR pathogens. Thus, the HTCS are always the focus of intensive cleaning in high-risk areas, especially when MDR-AB is epidemic or endemic. Considering that visual cleanliness does not always mean a microbiological cleanliness, previous studies showed that an invisible fluorescent marker is a much better strategy to improve environment cleaning, by quantitatively assessing cleaning and disinfecting practices on MRSA and VRE (16, 17). However, to our best knowledge, there is no report on this method used to evaluate impact of hospital environmental cleaning in China, especially for MDR-AB.

In this study, we utilized an invisible fluorescent marker, together with bacterial culture and identification, to evaluate the environmental cleaning on the HTCS within ICUs of Nanjing Drum Tower Hospital, and to explore the impact of environmental cleaning on the colonization and infection rates of MDR-AB in patients from 2013 to 2014.

Study design

The study was conducted within Nanjing Drum Tower Hospital, a 3,325-bed general tertiary care and university-affiliated teaching hospital in Nanjing, Jiangsu province, China.

The study design was shown in Figure 1. Briefly, the samples of patients hospitalized into in a 27-bed intensive care unit (ICU) in our hospital were continuously taken to monitor MDR-AB from 2013 to 2014. The bedrail, bedside table, injection pump button, monitor button, treatment vehicle and treatment table were chosen and referred to as HTCS, basing on hand contact frequency and easy-to-contact area of patients. The HTCS were marked by fluorescence labeling and continuously monitored for clearance level of fluorescence labeling and for contamination rate of MDR-AB, which was resistant against at least one agent in three or more of tested antimicrobial categories (18).

Labeling of fluorescence marks and determination of the clearance level

The fluorescence marks were drawn by a fluorescent pen (RUHOF), which uses a special nontoxic target solution. When exposed to black light, the marks emit fluorescence brightly. Noteworthily, the fluorescence marks are inconspicuous, dry rapidly on surfaces, remain environmentally stable for several weeks, resist dry abrasion, but could be easily removed by minimal abrasion with moistened cloth (17, 19). Fluorescence labeling was performed before cleaning (twice a day at 10 a.m. and 5 p.m.), one mark every square centimeter (Figure S1). The targets were evaluated following terminal cleaning after patients had occupied the room. Clearance level of fluorescence labeling was calculated by comparing the number of fluorescence marks before and after cleaning. Environment cleanliness was divided into cleaning (labeling clearance rate >80%) and contamination (labeling clearance rate < 80%).

Monitoring contamination of MDR-AB on the HTCS

To monitor contamination of MDR-AB on the HTCS of the ICUs, samples were accordingly taken for bacterial culture from each site of fluorescence labeling by a cotton swab moistened with saline, according to the Technical Specification for Disinfection of Hospital Disinfection Hygiene Standard issued by the Ministry of Health of China (http://www.biaozhun8.cn/biaozhun108760). Once A. baumannii was detected, antimicrobial susceptibility was further implemented to detect MDR-AB.

The colonization and infection rates of MDR-AB among inpatient in the ICU

Clinical samples including sputum, urine, blood, etc., from patients within the ICU during 2013-2014 were routinely taken and sent to the clinical microbiology laboratory for bacterial culture and susceptibility testing. The diagnostic criteria for colonization and infection referred to the criteria issued by the US CDC in 2008 (20). According to the international epidemiological quantitative statistical methods, the newly isolated multidrug-resistant bacteria per thousand hospitalization days was adopted as the quantitative statistical standard, that is, the detection or infection density of multidrug-resistant bacteria in a specific time range (Number of newly isolated multidrug-resistant bacteria infected or colonized new patients in a period/number of hospital days in a period).

Bacterial identification and antimicrobial susceptibility testing

Strains isolated were identified by ATB32E or Vitek-2 technology (Bio-Meriere, France). The susceptibility was determined by Kirby-Bauer method according to the guidelines of CLSI 2015. The tested antimicrobial agents were as follows: amikacin, ceftazidime, cefoperazone/sulbactam, imipenem, meropenem, piperacillin-tazobactam, cefepime, ticarcillin/clavulanate, ciprofloxacin, levofloxacin, sulfamethoxazole, minocycline and tigecycline. Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were used as the quality controls in parallel.

Pulsed field gel electrophoresis

When three or more MDR-AB were detected simultaneously in a short period of time, the genetic relatedness among those MDR-AB strains collected from the patients and the HTCS during the same period were further analyzed through pulsed field gel electrophoresis (PFGE) according to the protocol (21). Briefly. Fresh and pure bacterial cultures were embedded in agarose plugs and digested with proteinase K (20 mg/mL), followed by paI restriction endonuclease (TaKaRa, Dalian, Beijing, China). The standard strain Salmonella enterica serotype Braenderup H9812 digested with XbaI was used as a marker. The electrophoresis was performed in 0.5 × TBE buffer in a pulsed-field electrophoresis system (Chef Mapper; Bio-Rad Laboratories, Hercules, CA, USA), and the conditions were as follows: 14°C, 6 V/cm, switch angle 120°, switch ramp 5–20 s for 19 h. BioNumerics software version 7.6 (Applied Maths, Sint-Martens-Latem, Belgium) was used to analyze the PFGE banding patterns. A cut off of 80% was used to judge the relatedness of strains analyzed based on the tree constructed by the unweighted pair group method of averages and a position tolerance of 1.5% (20).

Statistical analysis

The correlation between the removal level of fluorescence labeling and colonization rates of MDR-AB, and the relationship between the colonization rates and infection rates of MDR-AB were analyzed by the Spearman correlation analysis through IBM SPSS Statistics 20. p< 0.05 was taken as statistically significant.

Clearance of fluorescence labeling

According to the hand contact frequency and easy-to-contact area of patients, the environmental surfaces in the ICU of our hospital, including bedrail, bedside table, injection pump button, monitor button, treatment vehicle and treatment table were chosen and referred to as HTCS. Then, fluorescence marks were labeled according to Figure S1 on these HTCS and continuously monitored for clearance level.

At the initial stage, the clearance rate of fluorescence labeling was comparatively low, only 21.9% (Table 1). Through training and strengthening supervision of cleaning workers, the total clearance rates of fluorescence labeling were greatly improved and finally reached up to 85.7% at the last quarter of 2013. However, with the frequent change and mobility of cleaning workers within ICU during 2014, the average clearance rate sharply decreased to less than 50%, even though frequent straining and education were implemented.

The contamination rate of MDR-AB on the HTCS within ICUs

To monitor the contamination level of MDR-AB on the HTCS, samples were collected for bacterial culture and identification. At the first quarter of 2013, 6 MDR-AB isolates were detected from HTCS. Coincidence with the increasing clearance rates of fluorescence labeling, the contamination rate of MDR-AB decreased remarkably, and no MDR-AB was identified. During the whole year of 2014, the average clearance rate of fluorescence labeling fell dramatically to less than 50%, MDR-AB were detected again on the HCTS. Overall, the contamination rate of MDR-AB on the HTCS was closely associated with the clearance rate (summarized in Table 1).

The colonization and infection rates of MDR-AB among inpatient within the ICU

To investigate the colonization and infection rates of MDR-AB among inpatient within the ICU, specimens from inpatient were collected for bacterial culture and identification. Totally, 126 and 168 non-replicative A. baumannii isolates were detected in 2013 and 2014, respectively, of which, 22.2% (28/126) in 2013 and 16% (27/168) were MDR-AB.

As shown in Table 1, in 2013, the hospital colonization rate of MDR-AB per Bed Days changed in the wake of the clearance level of fluorescence labeling. Spearman correlation analysis found a significant association between them (p=0.021). In addition, we found that the changes in the infection rates of MDR-AB were consistent with the ones in the colonization rates of inpatient within the ICU. However, we did not find a significant correlation between the colonization rates and the infection rates of MDR-AB.

Overall, the increased clearance rates of fluorescence marks lead to decreased contamination rates of MDR-AB on HTCS. And the colonization rates and infection rate of MDR-AB in inpatient also decreased correspondingly.

The genetic relatedness of the MDR-AB strains during the infection outbreak

In a month of 2014, 7 MDR-AB strains were isolated from sputum samples of 7 inpatients. Among them, 3 strains were associated with nosocomial infections (the

positive sputum culture was drawn >2 days after admission) (22), 4 strains with community infections (the positive sputum culture was drawn <2 days after admission). At the same time, 7 MDR-AB strains were isolated from the HTCS of these patients. According to the cutoff of 85%, all the MDR-AB displayed a genetic relatedness, indicating the existence of an epidemic clone (Figure 2).

In China, the largest developing country in the world, there are always much more patients and fewer beds even in the large Third-Class A General Hospitals. It is difficult to achieve isolation measures strictly, such as single-room for each patient or the same-room placement for the same multidrug-resistant bacterial infections or colonizers. Thus, the formulated practical measures and training for all kinds of medical personnel to strengthen environmental cleaning and disinfection are very important to prevent transmission of drug-resistant bacteria. Meanwhile, the economical and effective method for evaluation of environment cleaning to prevent hospital outbreaks is also urgently needed.

In this study, we utilized a fluorescence labeling method to systematically evaluate environment cleaning work for two consecutive years in Nanjing Drum Tower Hospital, a large tertiary hospital of Nanjing, southeast China. We focused on the relationship between cleanliness of the HTCS and colonization as well as infection of superbug MDR-AB within the ICU. In the initial stage of fluorescence labeling, the low clearance rate of fluorescent marks corresponded to a bad actual environmental cleaning, which means more than 50% of the HTCS that should be wiped were actually not cleaned. However, when the data were fed back to the management department of cleaners, the cleaning processes were optimized, due to more training and stricter supervision. These cleaning and disinfecting measures greatly improved cleaning level of the environment surfaces as shown by the obviously increased clearance rates of fluorescence labeling. However, with the frequent change and mobility of cleaning workers, the environment cleaning was greatly affected albeit regular training was still implemented, which emphasized the importance of the stability of cleaning workers within ICUs. Anyhow, our study showed that the fluorescence labeling method is suitable for national conditions in China, and worked very well to objectively assess environmental cleanliness and quantitative monitoring of educational intervention.

In addition, the consistence between the colonization rate and the infection rate indicated that the colonization of MDR-AB is the major resources of infections, and the correlation between the fluorescence clearance rates and colonization rates strongly underlined the importance of the environmental cleanness.

The close genetic relationship of the MDR-AB isolated from the environmental surfaces and specimens of patients provided a strong basis that the dissemination of the same clone strain among patients is positively correlated with the degree of environmental pollution in hospital wards, indicating the importance of the sanitation of hospital environment which has been repeatedly emphasized. Thus, strengthening the cleanness and disinfection of the HCTS and timely diagnosis and treatment of inpatients with MDR-AB infection or colonized patients is an effective way to prevent and control the prevalence and outbreak of MDR-AB within hospitals. More attention should be paid for the HTCS in hospital environment, since it played an increasingly media role in the transmission of pathogens in hospitals.

Outbreak of infections is mainly caused by an epidemic MDR-AB, which may easily colonize on the HCTS in hospitals. Fluorescence labeling is an effective way to objectively monitor the level of environmental cleanliness, which is in turn closely associated with MDR-AB colonization and infection of the inpatient within ICUs.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by the Nanjing Medical Science and technique Development Foundation (Grant no: QRX17059 and QRX17144), the Youth Fund of Jiangsu Province (Grant no. BK20170133).

Authors' contributions

Yang Li and Hai Ge implemented the fluorescent labeling, evaluation of fluorescent removal rates. Wanqing Zhou, Jie Zheng and Hui Zhou performed the bacterial identification, susceptibility testing, Wei Chen and Xiaoli Cao analyzed the data and revision on the manuscripts.

Acknowledgments

None

Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of Laboratory Medicine, Nanjing Drum Tower Hospital, the affiliated Hospital of Nanjing University Medical School, and the Clinical Research Center, the second hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003, China

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Table 1. the Number of fluorescent marks and clearance of high frequency contact sites as well as fluorescence clearance rate

HTCS	The number of fluorescent markers and removel	Time period
HTCS	The number of fluorescent markers and removel	1-3, 2013	4-6, 2013	7-9, 2013	10-12, 2013	1-3, 2014	4-6, 2014	7-9, 2014	10-12, 2014
Guardrail	Marker number	520	431	463	531	568	446	420	467
Guardrail	Scavenging number	102	139	377	489	283	186	175	173
Bedside table	Marker number	116	124	112	127	108	91	74	85
Bedside table	Scavenging number	23	89	100	115	75	47	44	31
Treatment vehicle	Marker number	132	97	144	105	87	96	111	93
Treatment vehicle	Scavenging number	54	73	124	87	39	39	57	55
Monitor button	Marker number	194	108	137	99	115	127	100	86
Monitor button	Scavenging number	31	43	87	76	47	53	41	26
Injection pump button	Marker number	191	84	154	116	152	110	242	131
Injection pump button	Scavenging number	58	27	119	84	70	31	73	48
Treatment table	Marker number	46	18	33	29	19	15	41	62
Treatment table	Scavenging number	1	7	28	12	5	6	17	21
total	Marker number	1199	862	1043	1007	1049	885	988	924
total	Scavenging number	269	378	835	863	519	362	407	354
Clearance rate (%)		21.9	43.9	80	85.7	49.5	40.9	41.2	38.3

Table 1. The clearance rates of fluorescent marks, the colonization rates of multidrug-resistant Acinetobacter baumannii and the infection rates of multidrug-resistant Acinetobacter baumannii of the inpatients within ICU

Time period	Fluorescent label clearance rates (%)	Bed Days	The number of MDRAB for colonization	The number of MDRAB for nosocomial infections	The daily colonization rates of MRDAB per thousand bed (‰)	The hospital infection rates of MDR-AB per thousand bed days (‰)
1-3, 2013	21.9	1761	29	13	16.47	7.38
4-6, 2013	43.9	1845	33	3	17.89	1.63
7-9, 2013	80	1542	14	7	9.08	4.54
10-12, 2013	85.7	1775	10	5	5.63	2.82
1-3, 2014	49.5	1972	18	3	9.13	1.52
4-6, 2014	40.9	1824	30	12	16.45	6.58
7-9, 2014	41.2	1779	15	5	8.43	2.81
10-12, 2014	38.3	1810	20	7	11.05	3.87

Impact of environmental cleaning on the colonization and infection rates of multidrug-resistant Acinetobacter baumannii in patients within the intensive care unit in a tertiary hospital

Status:

Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Conclusion

Declarations

References

Tables

Status:

Journal Publication

Version 1