|Year : 2018 | Volume
| Issue : 3 | Page : 147-152
Biofilm formation by carbapenem-resistant Enterobacteriaceae strains isolated from surveillance cultures in Intensive Care Unit patients: A significant problem
Ekadashi Rajni1, Vikas Rajpurohit2, Praveen Kumar Rathore3, Deep Shikhar3, PK Khatri3
1 Department of Microbiology, RUHS College of Medical Sciences, Jaipur, Rajasthan, India
2 Department of Anaesthesia, SNMC, Jodhpur, Rajasthan, India
3 Department of Microbiology, SNMC, Jodhpur, Rajasthan, India
|Date of Submission||12-Jun-2018|
|Date of Acceptance||07-Aug-2018|
|Date of Web Publication||24-Dec-2018|
Dr. Ekadashi Rajni
RUHS College of Medical Sciences, Jaipur, Rajasthan
Source of Support: None, Conflict of Interest: None
Background: Carbapenem-resistant Enterobacteriaceae (CRE) is a serious global public health threat. Antibiotic overuse, improper sanitation and unhygienic practices lead to large scale carriage and rapid spread. Biofilm formation by these strains can lead to far reaching consequences. Material and Method: This is a prospective hospital based study planned for a period of 3 months including all patients admitted to 14 bedded Trauma ICU of a tertiary care hospital in Rajasthan. Rectal swabs were collected from admitted patients and carriage of carbapenem resistant enterobacteriaceae looked for as per CDC guidelines. Screening of the Enterobacteriaceae colonies for carbapenemase production was done by Modified Hodge test. Carbapenem-resistant isolates were also tested for Metallobetalactamase production by phenotypic disc confirmatory test. Biofilm formation testing was done by Tissue culture plate method. Results: A total of 73 patients were screened and 27 CRE isolates were obtained, carriage rate being 37%. A high level of resistance was seen to aminoglycosides, fluoroquinolones and cephalosporins. 100% sensitivity was however seen to Colistin, Tigecycline and Fosfomycin. 5 out of 27 strains showed a positive MHT test. Metallo beta lactamase (MBL) production was seen in 21/25 strains as tested by meropenem and meropenem-edta discs. Of the 27 CRE isolates tested for biofilm production, 13 (48.15%) were found positive. Conclusion: The current study finds out the prevalence of CRE carriage and the biofilm forming potential of these strains among critically ill patients and stresses upon urgent need for stringent infection control measures.
Keywords: Biofilm, carbapenem-resistant Enterobacteriaceae, Intensive Care Unit, rectal swab, surveillance
|How to cite this article:|
Rajni E, Rajpurohit V, Rathore PK, Shikhar D, Khatri P K. Biofilm formation by carbapenem-resistant Enterobacteriaceae strains isolated from surveillance cultures in Intensive Care Unit patients: A significant problem. J Health Res Rev 2018;5:147-52
|How to cite this URL:|
Rajni E, Rajpurohit V, Rathore PK, Shikhar D, Khatri P K. Biofilm formation by carbapenem-resistant Enterobacteriaceae strains isolated from surveillance cultures in Intensive Care Unit patients: A significant problem. J Health Res Rev [serial online] 2018 [cited 2019 Dec 8];5:147-52. Available from: http://www.jhrr.org/text.asp?2018/5/3/147/248445
| Introduction|| |
Carbapenem-resistant Enterobacteriaceae (CRE) are emerging extremely drug-resistant pathogens. They are present in the community as well as in hospitals. These organisms are typically resistant to nearly all available antimicrobial agents and infections caused by them are associated with an increased risk of mortality. Antibiotic overuse and improper sanitation and hygienic practices lead to the large-scale carriage of these multidrug-resistant isolates and their rapid spread in the intestinal microbiota.,,
Clinical cultures done routinely in the laboratory detect only a small percentage of patients with CRE. Various studies have proven that asymptomatic carriers are at a higher risk of developing an invasive infection. Since these resistant bugs are hardy organisms, the carrier, and surrounding environment form a kind of ecological niche where these organisms survive and proliferate., There is also a likelihood of the colonized patient spreading the bugs, and hence, acting as a source of cross-infection for other inmates. The patient care items have been time and again proven as fomites in healthcare-associated outbreaks. Introduction of new and complex equipment in the Intensive Care Unit (ICU) which is difficult to disinfect only serve to worsen the scenario.
Such a carriage CRE strain if happens to be a biofilm producer can really worsen matters. Once they inhabit the environment, they are difficult to eradicate. The admitted patients being largely sick, elderly, or immunocompromised, need to be put on various devices such as central line, urinary catheter, and ventilator support. These resistant bacteria which the patient very innocuously carry in the gut can then colonize the device leading to device-associated infections.,,
There is a paucity of data on the prevalence of such organisms in our intensive care setup. Therefore, this prospective hospital-based pilot study was planned for 3 months to evaluate the carriage of CRE isolates from rectal swab collected from patients and also determine their biofilm forming ability.
| Materials and Methods|| |
This was a prospective hospital-based pilot study planned for 3 months, from January to March 2018 and included all the patients admitted to 14-bedded Trauma ICU of Mathura Das Mathur Hospital (MDMH), Jodhpur, who agreed to be a part of the study. Jodhpur is the second largest city in the Indian state of Rajasthan and officially the second metropolitan city of the state. MDMH is a tertiary level care hospital and caters to a large population from in and around Jodhpur. For conducting the CRE surveillance, rectal swab was collected from each patient on the day of their admission. A sterile swab was introduced approximately 1 inch into anal canal, rotated slowly for 10 s and put immediately in 5 ml of Trypticase soy broth to which a 10 μg of meropenem disc had already been added. This was incubated overnight at 37°C. The following day, incubated broth culture was vortexed, and 100 μl of the broth was subcultured on MacConkey agar plate and streaked for isolation. The growth of lactose fermenting colonies if any was looked for identification and susceptibility testing performed as per the standard guidelines. Screening of the Enterobacteriaceae colonies for carbapenemase production was done by modified Hodge test (MHT) as per the Clinical and Laboratory Standards Institute guidelines. Carbapenem-resistant isolates were also tested for metallobetalactamase (MBL) production by phenotypic disc confirmatory test.
Biofilm forming capacity was tested using tissue culture plate method as described by Christensen et al.
Risk factors of patients with and without CRE colonization were also analyzed. We have used t-test to calculate the significance of proportion and odds ratio (OR) to judge the strength of association of risk factors.
Ethical clearance for the study was obtained by Institutional Ethical Committee vide number No F1/Acad/MC/JU/18/5152.
All the samples were tested under Mukhyamantri Nishulk Jaanch Yojna which stipulates that all road traffic accident cases are investigated and treated free of cost.
| Results|| |
A total of 73 patients admitted to trauma ICU were screened over a period of 3 months. The most common cause of admission was a road traffic accident with resultant head injury. From the 73 patients included in the study, 27 CRE isolates were obtained, carriage rate being 37%. The average age was 41 and 30 years among colonized and noncolonized patients, respectively. The patients were mostly males [Table 1].
All the patients needed urinary catheterization. About 95% of the patients also needed ventilator support and hemodynamic monitoring through a central line. History of antibiotic usage was elicited from 14 (52%) of the CRE carriers and 30 (65%) noncolonized patients. History of the previous hospitalization was elicited from 13 (48%) to 27 (59%) of colonized and noncolonized patients, respectively. All the patients, except one, were initially healthy to begin with and had no history of any comorbidity. No significant correlation was found with the presence of comorbidity, history of previous hospitalization, or antibiotic usage [Table 2].
|Table 2: Distribution of patients with and without carbapenem-resistant Enterobacteriaceae colonization as per certain associates|
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Twenty-seven CRE isolates included 15 Klebsiella pneumoniae and 12 Escherichia More Details coli. [Table 3]. Sensitivity testing of these 27 CRE strains was done by Vitek. A high level of resistance was seen to aminoglycosides, fluoroquinolones, and cephalosporins. 100% sensitivity was, however, seen to colistin, tigecycline, and fosfomycin [Table 4].
|Table 4: Antimicrobial sensitivity pattern of carbapenem-resistant Enterobacteriaceae strains|
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Five out of 27 strains showed a positive MHT test. MBL production was seen in 21/25 strains as tested by meropenem and meropenem-ethylenediaminetetraacetic acid (EDTA) discs.
Of the 27 CRE isolates tested for biofilm production, 13 (48.15%) were found positive. These included 4 (14.82%) strong biofilm producers and 9 (33.33%) moderate ones. Fourteen (51.85%) CRE strains were biofilm negative. Further break up revealed that 8 (53.33%) out of 15 strains of Klebsiella and 5 (41.67%) out of 12 strains of E. coli were biofilm producers [Table 5].
|Table 5: Biofilm production among carbapenem-resistant Enterobacteriaceae strains|
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| Discussion|| |
Since the early days when Antonie van Leeuwenhoek, first observed “animalcule” on his tooth surface using a simple microscope, a lot of research has gone into the biofilms. Costerton coined the term in 1978 and explained the benefits that the microorganisms derive because of this ecological niche. Biofilm forming capacity has been studied extensively in various clinical isolates such as Staphylococcus aureus, Coagulase-negative Staphylococcus and Acinetobacter baumanni and has been linked to many hospital-acquired infections and outbreaks. There are also ample data to suggest increased virulence and antimicrobial resistance among the biofilm producers. There is, however, negligible data on the biofilm formation in the resistant bugs found in surveillance studies and how they may influence the clinical condition of the patient.
CRE has become a major global health threat. Whether isolated from clinical samples or surveillance cultures, their importance in determining the outcome of the patient cannot be understated. They are resistant to even the last resort antibiotics leading to increased morbidity and mortality.,,, These are troublesome pathogens and once they inhabit the ICU ecosystem are difficult to eradicate. Contamination of the inanimate surfaces around the patient with these bugs serve as a constant reservoir of infection for the patient who is a carrier and also to whom who share the hospital milieu. There is an enhanced risk of their transmission during procedures. They target the vulnerable patient population and lead to various hospital-acquired infections. Biofilm forming potential causes this group of bacteria to become adherent to each other and get embedded within a matrix of extracellular polymeric substance known as exopolysaccharide. This creates a protective environment causing difficulty in antibiotic penetration and provides protection against insults such as dehydration and limited nutrient availability. The biofilm continues to shed microcolonies of these resistant organisms into the patient thereby perpetuating infection. If stringent infection control measures are not followed, and there is cross-infection between other patients and outbreaks become imminent.,, Considering these facts, it is important that surveillance culture is done on patients, at least in high-risk settings.,,
The current study was carried out to know the prevalence of CRE in rectal swabs in the patients admitted to Trauma ICU of a tertiary care hospital and to study their antimicrobial sensitivity pattern and biofilm forming capacity. To the best of our knowledge, this is the first study to assess biofilm production by CRE isolates from surveillance cultures.
A total of 27 CRE isolates were obtained. MHT was positive in five strains signifying carbapenemase production. Since MHT is known to be highly sensitive for detection of K. pneumoniae carbapenemase (KPC) production, it is safe to presume that some other mechanisms were also responsible for resultant carbapenem resistance. Twenty-one strains showed ≥7 mm increase in the zone of inhibition when tested by meropenem and meropenem-EDTA disc combination, thus signifying MBL production. More conclusive data would need molecular studies which could not be done because of lack of infrastructure.
Out of 73 cases admitted to Trauma ICU, 27 (37%) CRE isolates were obtained, that is, about one-third of the patients have been detected with the colonization of CRE. This percentage of CRE colonization was highly significant (t = 6.72, P <.01) as well. Of these 27 CRE isolates tested for biofilm production, 13 (48.15%) were found positive. These included 4 (14.82%) strong biofilm producers and 9 (33.33%) moderate ones. The mean age of patients found colonized with CRE was 41.52, while the mean age of patients not colonized with CRE was 30.61 years. The difference in age of patients (pts.) colonized with CRE and not colonized with CRE was also significant (t = 3.008, P < 0.01). [Table 2] presents the analysis of some associates of CRE colonization. Data in Row 1 of this table yield significant OR = 3.88, P = 0.009 with respect to risk of colonization of CRE in older pts. (Age 40+).
The risk of mortality among pts with CRE colonization is found a bit higher (OR = 1.25, P = 0.478) as read from data row 5 of this table. The percentage of mortality among pts with CRE is also found higher than the percentage of mortality among pts without CRE colonization. This then suggests for better care for CRE colonized pts, in particular in the age group of 40+. The other associates, namely patients on ventilator, pts with tracheostomy, or pts with the previous history of antibiotic use did not show OR >1 as read from data rows 2, 3, and, 4 of this table.
Various studies have found that being on ventilator support is a significant risk factor for CRE colonization.,,,,, However, in our study, this is not the case. This may be due to the lesser number of study participants or the duration of the study. However, even this small study makes it clear that in ICUs, the percentage of pts with CRE colonization is high, particularly in older age group patients and mortality is also more in patients with CRE colonization.
In a study conducted in a tertiary care teaching hospital in Delhi, out of the 242 isolates tested from patients attending the outpatient department, 24 (9.9%) demonstrated carbapenemase activity. Lower rates found in this study may be explained by the fact that it was done on outpatients. The duration of hospital stay and antibiotic use has been shown to increase the risk of fecal carriage of CRE in many studies., Twelve (29%) E coli and 8 (38%) Klebsiella spp. isolated from rectal swabs were found to be positive for MBL production in a study by Aggarwal et al., Mittal et al. have reported fecal carriage of carbapenemase-producing Enterobacteriaceae (CPE) in 11% and 22% in ICU patients on the day 1 and day 4, respectively.
On a global level, gastrointestinal carriage rate of CRE has been shown to be highly variable. The prevalence of CPE carriers admitted to ICUs in 11 acute care Spanish hospitals was 1.6%–2.4%. In a Korean study, fecal carriage of CRE was found in 0.3% patients on admission. None of the CRE isolates was a carbapenemase producer. During a prospective observational study conducted on all patients admitted to an ICU in a hospital in Greece, 12.8% of patients were found to be colonized. In a study conducted in a long-term care facility in New York, CRE carriage was noticed in 18.9% of patients. Percentages of patients colonized on ICU admission have been reported to be around 5%–7% in Israel and 11% in Mexico., Almost negligible rates have been found in Germany and Algeria.,
None of the studies have explored the biofilm forming capacity of these strains. During our study period, clinical samples were also collected from patients suspected to have a nosocomial infection. Interestingly, one CRE carrier developed central line-associated bloodstream infection, and the central line tip culture grew a Klebsiella strain which had a similar antibiotic sensitivity profile as that of Klebsiella isolated from the rectal swab. Likewise, another patient developed catheter-associated urinary tract infection, and the urine culture grew Klebsiella which again had a sensitivity profile similar to that isolated from rectal swab. Both these strains were strong biofilm producers. It is quite probable that these were the same carriage strains that caused the nosocomial infections. Sequencing of both the strains could solve this dilemma.
If a patient is found to be colonized with these notorious bugs, a battery of precautions needs to be followed. As far as possible, single room accommodation should be provided. In a resource-limited country like ours, where this is not possible, geographic, and personnel cohorting should be done. Number of visitors in the room should be minimized, and patients may be advised to remain in the room except for emergency procedures. Transfer between facilities should only be done if medically necessary. Strict compliance to infection control practices such as hand hygiene and religious use of personal protective equipment are important tools. Device use should be minimized, and antibiotics should be used prudently. Once a device-associated infection occurs in a colonized patient, it may not be possible to salvage the device and removal becomes necessary.,,,
CRE transmissions have been reported from various reusable flexible endoscopic instruments. Enhanced training and monitoring of processing of reusable items are required. There are reports on the presence of biofilm containing viable multi-resistant organisms despite terminal cleaning on clinical surfaces in ICU. Cleaning, high-level disinfection, and drying need to be done in strict compliance with hospital policy and manufacturer's instructions.,,
Our research is limited by short-time period and small sample size. Furthermore, due to infrastructure limitations, molecular analysis of CRE strains could not be done. The current pilot study, however, manages to find the prevalence of CRE carriage among critically ill patients and also show that a good number of them are also biofilm producers. Thus, it serves to sensitize the health-care community about the existence of the problem and the impress on the strong need for stringent infection control measures.
| Conclusion|| |
This pilot study will guide clinicians and clinical microbiologists about colonization of such multidrug-resistant strains in the human gastrointestinal tract. Early detection of asymptomatic rectal carriage of CRE and their biofilm forming potential is important for infection control purposes.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]