- Email: [email protected]
Faecal carriage rate of extended-spectrum β-lactamase-producing Enterobacteriaceae in hospitalised patients and healthy asymptomatic individuals coming for health check-up
Journal of Global Antimicrobial Resistance 6 (2016) 150–153
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Short Communication
Faecal carriage rate of extended-spectrum b-lactamase-producing Enterobacteriaceae in hospitalised patients and healthy asymptomatic individuals coming for health check-up Rachana Babu a, Anil Kumar a,*, Shamsul Karim a, Sruthi Warrier b, Suresh G. Nair c, Sanjeev K. Singh d, Raja Biswas b a
Department of Microbiology, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi 682041, Kerala, India Center for Nanoscience & Molecular Medicine, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi, Kerala, India c Department of Anesthesia and Critical Care, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi, Kerala, India d Department of Infection Control, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi, Kerala, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 January 2016 Received in revised form 4 May 2016 Accepted 8 May 2016
The prevalence of extended-spectrum b-lactamase-producing Enterobacteriaceae (ESBL-PE) in hospitalised and community patients is of significant public health concern. The aim of this study was to estimate the faecal carriage rate of ESBL-PE in hospitalised patients and healthy asymptomatic individuals coming for health check-up. Non-repetitive, consecutive stool samples from 480 adults (260 healthy individuals and 220 hospitalised patients) aged 18 years from November 2011 to July 2013 were screened using MacConkey agar supplemented with ceftazidime. All screen-positive isolates were identified to species level and were tested for ESBL production. Representative ESBL-PE isolates were subjected to susceptibility testing and multiplex ESBL PCR. The faecal carriage rate of ESBL-PE was found to be 62.7% among hospitalised patients and 33.8% among healthy asymptomatic individuals. The most common ESBL-PE was Escherichia coli (70.3% and 78.4% in hospitalised patients and healthy individuals, respectively), followed by Klebsiella pneumoniae (26.8% and 17.0%). ESBL-PE showed the highest sensitivity to carbapenems (85% and 100%, respectively), followed by amikacin (67.2% and 98%), cefoperazone/sulbactam (27.8% and 88.2%) and piperacillin/tazobactam (18% and 74.5%). Ciprofloxacin exhibited a high level of resistance among both groups. Molecular analysis for ESBL genes showed a predominance of the CTX-M gene. In conclusion, the faecal carriage rate of ESBL-PE among hospitalised patients was almost double that of healthy individuals. Carriage of carbapenem-resistant isolates is emerging among hospitalised patients. The spread of these organisms in the community merits radical measures to improve sanitation and implement antibiotic stewardship. ß 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: ESBL Escherichia coli Faecal carriage Carbapenem
1. Introduction The high prevalence of extended-spectrum b-lactamaseproducing Enterobacteriaceae (ESBL-PE) in developing countries has been attributed to the availability of over-the-counter antibiotics without prescription, widespread self-treatment, overcrowding of hospitals, the absence of antibiotic prescription guidelines, poor infection control practices and poor sanitation [1]. The problem is so wide spread that even travel to the Indian
* Corresponding author. Tel.: +91 484 280 1234x8015; fax: +91 484 280 2020. E-mail address: [email protected] (A. Kumar).
subcontinent has been associated with intestinal acquisition of ESBL-PE [2]. High intestinal carriage of ESBL-PE among hospitalised patients is well documented and is associated with a high risk of developing self- and cross-infections [3]. Studies have also traced the origin of uropathogenic ESBL-PE to the digestive tract of the same patient [3]. Apart from being important nosocomial pathogens, ESBL-PE are being increasingly reported from community-acquired infections owing to selection pressure from the widespread use of third-generation cephalosporins and fluoroquinolones [4]. Previous studies have documented 2–5 years of ESBL-PE digestive carriage [4]. Although there are many prospective longitudinal studies on faecal carriage of ESBL-PE among hospitalised patients, very few
http://dx.doi.org/10.1016/j.jgar.2016.05.007 2213-7165/ß 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
R. Babu et al. / Journal of Global Antimicrobial Resistance 6 (2016) 150–153
studies have investigated their prevalence in healthy asymptomatic individuals in the community [5]. This study aimed to determine the faecal carriage rate of ESBLPE among hospitalised patients and healthy asymptomatic individuals who come for general health check-up. 2. Materials and methods 2.1. Study setting This study was conducted in the Department of Microbiology at Amrita Institute of Medical Sciences (Kochi, India) from November 2011 to July 2013. Non-repetitive, consecutive stool samples from adults aged 18 years were collected after obtaining informed consent and were processed within 24 h of sampling. Among the 480 samples analysed, 260 were from healthy individuals who came for comprehensive healthcare check-up and 220 were from hospitalised patients admitted for >72 h in the hospital. Screening for ESBL-PE was carried out using MacConkey agar plates supplemented with 1 mg/mL ceftazidime. Phenotypic confirmation of ESBL was done for one isolate of each morphotype from each patient by the disk potentiation method. Klebsiella pneumoniae ATCC 700603 (positive control) and Escherichia coli ATCC 25922 (negative control) were used for validation. Antibiotic susceptibility testing of representative ESBL-PE isolates was performed by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion method [6]. 2.2. PCR detection of extended-spectrum b-lactamases (ESBLs) Genomic DNA was extracted by boiling and was used as the template for multiplex PCR with appropriate primers for blaTEM, blaSHV and blaCTX-M-1, -2 and -9 ESBL types using cycling conditions as described previously [5]. DNA amplification by PCR yielded fragments of ca. 550 bp for CTX-M, 867 bp for TEM and 392 bp for SHV. 2.3. Statistical analysis Associations between different underlying conditions and hospitalisation with faecal carriage of ESBL-PE were analysed using x2 and Fisher’s exact tests. Pair-wise analysis was conducted using IBM SPSS Statistics for Windows v.20.0 (IBM Corp., Armonk, NY). A P-value of <0.05 was considered statistically significant in all cases. 2.4. Ethical clearance This study was approved by the Institutional Ethics Committee of Amrita Institute of Medical Sciences. Informed consent was obtained from all of the participants before enrolment. 3. Results Among the 480 individuals screened for ESBL-PE, 260 were asymptomatic healthy individuals who came for health check-up and 220 were hospitalised patients admitted for >72 h in the hospital. Both groups had a preponderance of males [69.6% (181/260) among healthy individuals and 61.4% (135/220) among hospitalised patients]. The mean patient age was 45.5 years and 52.4 years for healthy individuals and hospitalised patients, respectively. Of the 220 hospitalised patients screened, the various underlying conditions (patients could have more than one underlying condition) were diabetes mellitus (63; 28.6%), coronary artery disease (23; 10.5%), malignancy (46; 20.9%), gastrointestinal
151
Table 1 Faecal carriage rate of extended-spectrum b-lactamase-producing Enterobacteriaceae among hospitalised patients (n = 220) and healthy individuals (n = 260). Hospitalised patients [n (%)]
Strain Escherichia coli Klebsiella pneumoniae Others Total
Healthy individuals [n (%)]
97 (44.1) 37 (16.8) 4 (1.8)
69 (26.5) 15 (5.8) 4 (1.5)
138 (62.7)
88 (33.8)
disorders (99; 45.0%), transplantation (3; 1.4%) and renal disease (26; 11.8). Statistical analysis of hospitalised patients with respect to different underlying diseases for faecal carriage of ESBL-PE showed none of them to be significant. However, when hospitalised patients were compared with healthy individuals, the former had a significant association (P < 0.001). All hospitalised patients had varying degrees of exposure to broad-spectrum antibiotics such as third-generation cephalosporins, fluoroquinolones, aminoglycosides and carbapenems. The mean duration of hospital stay in these patients was 16.23 days, and of these 30 patients died during the course of their treatment. The overall faecal carriage rate of ESBL-PE was 62.7% (138/220) among hospitalised patients and 33.8% (88/260) in healthy asymptomatic individuals. The different ESBL-PE species isolated are shown in Table 1. Antibiotic susceptibility testing of 51 representative isolates from healthy individuals and 61 isolates from hospitalised patients revealed a high degree of resistance in the latter. Cross-resistance to fluoroquinolones was the highest in both groups. Genotypic characterisation of representative ESBL-PE isolates (51 from healthy individuals and 53 from hospitalised patients) revealed a predominance of CTX-M genes (77–88%) (Table 2). The various combinations in which the ESBL genes (TEM, SHV and CTXM) were present in these isolates are given in Table 2. The presence of all the three ESBL genes was more often seen in Klebsiella spp. compared with E. coli. The presence of CTX-M and its association with cross-resistance to other antibiotics was analysed. Resistance to ciprofloxacin was the highest, with 81% in isolates from hospitalised patients and 60% in isolates from healthy individuals. The other notable feature was the emergence of carbapenem resistance (14.8%; 9/61) in ESBL-PE isolates from hospitalised patients and their absence in isolates from healthy individuals. The actual carbapenem resistance rate in hospitalised patients is expected to be ca. 20–30% considering the fact that only 61 isolates were eventually tested. Carbapenemresistant isolates were predominantly K. pneumoniae (5/9), followed by E. coli (2/9) and Enterobacter spp. (2/9). The majority (6/9) of these patients were also being treated with carbapenems.
Table 2 Genotypic distribution of extended-spectrum b-lactamase (ESBL) genes in ESBLproducing Enterobacteriaceae isolates from hospitalised patients and healthy individuals (n = 53/51). ESBL gene
Escherichia Klebsiella Enterobacter coli (n = 34/37) pneumoniae (n = 17/13) spp. (n = 2/1)
CTX-M SHV TEM CTX-M and SHV SHV and TEM CTX-M and TEM CTX-M, SHV and TEM None
16/26 1/0 3/0
9/5 2/0 3/4
3/4
4/2 1/1 1/2 7/2 1/1
1/0 1/0
152
R. Babu et al. / Journal of Global Antimicrobial Resistance 6 (2016) 150–153
Table 3 Comparison of faecal carriage rates of extended-spectrum b-lactamase-producing Enterobacteriaceae (ESBL-PE) from different studies in India. Author/year
Location
Deshpande et al., 2012 [7] Rodrigues et al., 2005 [8] Mathai et al., 2015 (n = 22) [5] Vandana et al., 2010 [9] Kothari et al., 2013 (n = 27) [10] Sarma and Ahmed, 2010 [11] Azim et al., 2010 [12] Present study, 2013 (n = 106)
Mumbai Mumbai Vellore Manipal New Delhi Guwahati Lucknow Kochi
Faecal carriage rate of ESBL-PE (%)
ESBL gene rate (%)
Hospitalised patients
Healthy individuals
CTX-M
SHV
TEM
23.9 11 19
ND ND 95.5 ND 82.5 ND ND 83
ND ND 9 ND ND ND ND 20
ND ND 68 ND ND ND ND 34
18.5 20.6 26 92 62.7
33.8
ND, not done.
4. Discussion Faecal carriage of ESBL-PE is considered to be a risk factor for infection with these bacteria. Previous studies have documented high rates of infections such as urinary tract infection due to ESBLPE in patients who are also intestinal carriers of these pathogens [3]. Faecal carriage rates also vary with geographical location owing to factors such as hygiene, sanitation, availability of antibiotics etc. playing a major role in the dissemination of these bacteria. Previous Indian studies have reported faecal carriage rates ranging from 18% to 92% among hospitalised patients and from 11% to 24% among healthy individuals (Table 3) [5,7–12]. The present study reports a very high rate (62.7%) of faecal carriage among hospitalised patients, but this is still less than the 92% reported by Azim et al. from Lucknow, India [12]. The high rates can be explained by the fact that most of the patients had chronic illness and had undergone multiple admissions and courses of antibiotics. Among the healthy individuals, the faecal carriage rate of ESBL-PE was 33.8%, which is one of the highest reported in the literature. An identical study by Rodrigues et al. a decade earlier among healthy executives in Mumbai showed a rate of 11% [8]. One reason for such high carrier rates could be the urban nature of the population where prior use of antibiotics in the last 3 years cannot be discounted. Moreover, prior exposure to thirdgeneration cephalosporins and fluoroquinolones is not a prerequisite for acquiring ESBL-PE, as Mathai et al. reported a faecal carriage rate of 19% among an antibiotic-naı¨ve healthy tribal population with no access to healthcare facilities [5]. In 2012, Luvsansharav et al. reported an alarmingly high rate of 65.7% of CTX-M-positive ESBL-PE in a rural Thai community [13]. Close contact with colonisers and patients coupled with exposure to common sources such as contaminated food and water and poor sanitation have been suggested for their spread in the community [14]. The emergence of carbapenem-resistant Enterobacteriaceae isolates (14.8%) among hospitalised patients was also expected due to the widespread use of carbapenems for treating ESBL-associated infections. A study from Pakistan has already reported 18.5% faecal carriage of NDM-1 carbapenemase-producing isolates among hospitalised patients [15]. A recent report from Bangladesh also reported NDM-1 faecal carriage in patients with diarrhoea [16]. The total absence of carbapenem resistance among healthy individuals is reassuring, but with reports of isolation of carbapenem-resistant isolates from the environment it is only a matter of time before we start encountering intestinal colonisation by these isolates [17]. The predominance of CTX-M ESBL genes is also along the expected lines, as previous studies have reported CTX-M-15 to be the most common ESBL gene among Indian isolates (Table 3) [5,18,19]. The high degree of cross-resistance to fluoroquinolones among CTX-M-positive ESBL-PE can be explained by the cotransfer of plasmid-mediated qnr genes [18].
Therefore, it is not surprising that the first report describing CTX-M-15 and co-transfer of the qnrB plasmid-mediated gene along with CTX-M-15 were from Indian isolates [18]. CTX-M alleles also accounted for >90% of ESBLs among community ESBL-PE worldwide [1]. Historically, faecal carriage rates in Europe have rarely exceeded 10% in the community; in contrast, Thailand has reported rates as high as 69% [13]. Many European studies have documented high rates of acquisition of ESBL-PE associated with travel to Southeast Asia, Pakistan and the Middle East [2]. Acquisition rates as high as 73% have been reported in German travellers returning from India [2]. Studies on the clonality of ESBLproducing E. coli have shown the presence of diverse clones in the community. However, the presence of ST131 clone in India, Pakistan and Madagascar has been reported previously [20]. The dramatic increase in faecal carriage rates of ESBL-PE worldwide has been attributed to the efficient transfer of the plasmid-mediated blaESBL gene between Gram-negative bacteria. In a background of poor sanitation, ESBL-producing faecal coliforms (E. coli) can spread like wild fire leading to asymptomatic intestinal colonisation of the whole community, which then acts as a reservoir and subsequently leading to community-acquired infections in susceptible individuals. Despite the growing evidence on colonisers acting as a reservoir for nosocomial infections, routine screening of patients before admission is not recommended. Standard contact precautions and good infection control practices are all that is recommended for such patients. 5. Conclusion The faecal carriage rate of ESBL-PE among hospitalised patients was almost double that of healthy individuals. Carriage of carbapenem-resistant isolates is emerging among hospitalised patients and is a threat to the future of antibiotic chemotherapy. The high burden of ESBL-PE among healthy asymptomatic individuals has significant public health implications, including the treatment of community-acquired infections. The developing world, especially India, is staring at a pandemic of ESBL-PE. CTX-Mproducing E. coli are true community ESBL and their dissemination into the water supply and food chain needs to be prevented. The spread of these organisms in the community merits radical measures to improve sanitation and implement antibiotic stewardship. The major limitations of this study were that susceptibility testing, clonal typing for ST131 and genotyping for all ESBLproducing and carbapenem-resistant isolates were not performed. In the absence of sequencing, mere amplification of SHV and TEM genes may not necessarily mean ESBL production. Funding None.
R. Babu et al. / Journal of Global Antimicrobial Resistance 6 (2016) 150–153
Competing interests None declared. Ethical approval This study was approved by the Institutional Ethics Committee of Amrita Institute of Medical Sciences (Kochi, India). Informed consent was obtained from all of the participants before enrolment.
[10]
[11]
[12]
[13]
References [1] Woerther PL, Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extended-spectrum b-lactamases in the community: toward the globalization of CTX-M. Clin Microbiol Rev 2013;26:744–58. [2] Lu¨bbert C, Straube L, Stein C, Makarewicz O, Schubert S, Mo¨ssner J, et al. Colonization with extended-spectrum b-lactamase-producing and carbapenemase-producing Enterobacteriaceae in international travelers returning to Germany. Int J Med Microbiol 2015;305:148–56. [3] Tan BH, Tan AL. Community-acquired bacteremia with an ESBL-producing Escherichia coli strain—a case report. Int J Infect Dis 2002;6:318–9. [4] Arpin C, Dubois V, Maugein J, Jullin J, Dutilh B, Brochet JP, et al. Clinical and molecular analysis of extended-spectrum b-lactamase-producing enterobacteria in the community setting. J Clin Microbiol 2005;43:5048–54. [5] Mathai D, Kumar VA, Paul B, Sugumar M, John KR, Manoharan A, et al. Fecal carriage rates of extended-spectrum b-lactamase-producing Escherichia coli among antibiotic naive healthy human volunteers. Microb Drug Resist 2015;21:59–64. [6] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; approved standard—ninth edition. Document M2-A9. Wayne, PA: CLSI; 2013. [7] Deshpande P, Vadwai V, Shetty A, Dalal R, Soman R, Rodrigues C. No NDM-1 carriage in healthy persons from Mumbai: reassuring for now. J Antimicrob Chemother 2012;67:1046–7. [8] Rodrigues C, Shukla U, Jog S, Mehta A. Extended-spectrum b-lactamaseproducing flora in healthy persons. Emerg Infect Dis 2005;11:981–2. [9] Vandana KE, Varghese G, Krishna S, Mukhopadhyay C, Kamath A, Ajith V. Screening at admission for carrier prevalence of multidrug-resistant organisms
[14]
[15]
[16]
[17]
[18]
[19]
[20]
153
in resource-constrained settings: a hospital-based observational study. J Hosp Infect 2010;76:180–1. Kothari C, Gaind R, Singh LC, Sinha A, Kumari V, Arya S, et al. Community acquisition of b-lactamase producing Enterobacteriaceae in neonatal gut. BMC Microbiol 2013;13:136. Sarma JB, Ahmed GU. Prevalence and risk factors for colonisation with extended spectrum b-lactamase producing Enterobacteriaceae vis-a`-vis usage of antimicrobials. Indian J Med Microbiol 2010;28:217–20. Azim A, Dwivedi M, Rao PB, Baronia AK, Singh RK, Prasad KN, et al. Epidemiology of bacterial colonization at intensive care unit admission with emphasis on extended-spectrum b-lactamase- and metallo-b-lactamase-producing Gram-negative bacteria—an Indian experience. J Med Microbiol 2010;59: 955–60. Luvsansharav UO, Hirai I, Nakata A, Imura K, Yamauchi K, Niki M, et al. Prevalence of and risk factors associated with faecal carriage of CTX-M b-lactamase-producing Enterobacteriaceae in rural Thai communities. J Antimicrob Chemother 2012;67:1769–74. Valverde A, Grill F, Coque TM, Pintado V, Baquero F, Canto´n R, et al. High rate of intestinal colonization with extended-spectrum-b-lactamase-producing organisms in household contacts of infected community patients. J Clin Microbiol 2008;46:2796–9. Day KM, Ali S, Mirza IA, Sidjabat HE, Silvey A, Lanyon CV, et al. Prevalence and molecular characterization of Enterobacteriaceae producing NDM-1 carbapenemase at a military hospital in Pakistan and evaluation of two chromogenic media. Diagn Microbiol Infect Dis 2013;75:187–91. Islam MA, Nabi A, Rahman M, Islam M, Ahmed D, Faruque AS, et al. Prevalence of faecal carriage of NDM-1-producing bacteria among patients with diarrhoea in Bangladesh. J Med Microbiol 2014;63:620–2. Walsh TR, Weeks J, Livermore DM, Toleman MA. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 2011;11: 355–62. Jacoby GA, Walsh KE, Mills DM, Walker VJ, Oh H, Robicsek A, et al. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother 2006;50:1178–82. Muzaheed Y, Doi Y, Adams-Haduch JM, Shivannavar CT, Paterson DL, Gaddad SM. Faecal carriage of CTX-M-15-producing Klebsiella pneumoniae in patients with acute gastroenteritis. Indian J Med Res 2009;129:599–602. Herindrainy P, Randrianirina F, Ratovoson R, Ratsima Hariniana E, Buisson Y, Genel N, et al. Rectal carriage of extended-spectrum b-lactamase-producing Gram-negative bacilli in community settings in Madagascar. PLoS ONE 2011;6:e22738.
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Short Communication
Faecal carriage rate of extended-spectrum b-lactamase-producing Enterobacteriaceae in hospitalised patients and healthy asymptomatic individuals coming for health check-up Rachana Babu a, Anil Kumar a,*, Shamsul Karim a, Sruthi Warrier b, Suresh G. Nair c, Sanjeev K. Singh d, Raja Biswas b a
Department of Microbiology, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi 682041, Kerala, India Center for Nanoscience & Molecular Medicine, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi, Kerala, India c Department of Anesthesia and Critical Care, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi, Kerala, India d Department of Infection Control, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Ponekkara, Kochi, Kerala, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 January 2016 Received in revised form 4 May 2016 Accepted 8 May 2016
The prevalence of extended-spectrum b-lactamase-producing Enterobacteriaceae (ESBL-PE) in hospitalised and community patients is of significant public health concern. The aim of this study was to estimate the faecal carriage rate of ESBL-PE in hospitalised patients and healthy asymptomatic individuals coming for health check-up. Non-repetitive, consecutive stool samples from 480 adults (260 healthy individuals and 220 hospitalised patients) aged 18 years from November 2011 to July 2013 were screened using MacConkey agar supplemented with ceftazidime. All screen-positive isolates were identified to species level and were tested for ESBL production. Representative ESBL-PE isolates were subjected to susceptibility testing and multiplex ESBL PCR. The faecal carriage rate of ESBL-PE was found to be 62.7% among hospitalised patients and 33.8% among healthy asymptomatic individuals. The most common ESBL-PE was Escherichia coli (70.3% and 78.4% in hospitalised patients and healthy individuals, respectively), followed by Klebsiella pneumoniae (26.8% and 17.0%). ESBL-PE showed the highest sensitivity to carbapenems (85% and 100%, respectively), followed by amikacin (67.2% and 98%), cefoperazone/sulbactam (27.8% and 88.2%) and piperacillin/tazobactam (18% and 74.5%). Ciprofloxacin exhibited a high level of resistance among both groups. Molecular analysis for ESBL genes showed a predominance of the CTX-M gene. In conclusion, the faecal carriage rate of ESBL-PE among hospitalised patients was almost double that of healthy individuals. Carriage of carbapenem-resistant isolates is emerging among hospitalised patients. The spread of these organisms in the community merits radical measures to improve sanitation and implement antibiotic stewardship. ß 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: ESBL Escherichia coli Faecal carriage Carbapenem
1. Introduction The high prevalence of extended-spectrum b-lactamaseproducing Enterobacteriaceae (ESBL-PE) in developing countries has been attributed to the availability of over-the-counter antibiotics without prescription, widespread self-treatment, overcrowding of hospitals, the absence of antibiotic prescription guidelines, poor infection control practices and poor sanitation [1]. The problem is so wide spread that even travel to the Indian
* Corresponding author. Tel.: +91 484 280 1234x8015; fax: +91 484 280 2020. E-mail address: [email protected] (A. Kumar).
subcontinent has been associated with intestinal acquisition of ESBL-PE [2]. High intestinal carriage of ESBL-PE among hospitalised patients is well documented and is associated with a high risk of developing self- and cross-infections [3]. Studies have also traced the origin of uropathogenic ESBL-PE to the digestive tract of the same patient [3]. Apart from being important nosocomial pathogens, ESBL-PE are being increasingly reported from community-acquired infections owing to selection pressure from the widespread use of third-generation cephalosporins and fluoroquinolones [4]. Previous studies have documented 2–5 years of ESBL-PE digestive carriage [4]. Although there are many prospective longitudinal studies on faecal carriage of ESBL-PE among hospitalised patients, very few
http://dx.doi.org/10.1016/j.jgar.2016.05.007 2213-7165/ß 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
R. Babu et al. / Journal of Global Antimicrobial Resistance 6 (2016) 150–153
studies have investigated their prevalence in healthy asymptomatic individuals in the community [5]. This study aimed to determine the faecal carriage rate of ESBLPE among hospitalised patients and healthy asymptomatic individuals who come for general health check-up. 2. Materials and methods 2.1. Study setting This study was conducted in the Department of Microbiology at Amrita Institute of Medical Sciences (Kochi, India) from November 2011 to July 2013. Non-repetitive, consecutive stool samples from adults aged 18 years were collected after obtaining informed consent and were processed within 24 h of sampling. Among the 480 samples analysed, 260 were from healthy individuals who came for comprehensive healthcare check-up and 220 were from hospitalised patients admitted for >72 h in the hospital. Screening for ESBL-PE was carried out using MacConkey agar plates supplemented with 1 mg/mL ceftazidime. Phenotypic confirmation of ESBL was done for one isolate of each morphotype from each patient by the disk potentiation method. Klebsiella pneumoniae ATCC 700603 (positive control) and Escherichia coli ATCC 25922 (negative control) were used for validation. Antibiotic susceptibility testing of representative ESBL-PE isolates was performed by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion method [6]. 2.2. PCR detection of extended-spectrum b-lactamases (ESBLs) Genomic DNA was extracted by boiling and was used as the template for multiplex PCR with appropriate primers for blaTEM, blaSHV and blaCTX-M-1, -2 and -9 ESBL types using cycling conditions as described previously [5]. DNA amplification by PCR yielded fragments of ca. 550 bp for CTX-M, 867 bp for TEM and 392 bp for SHV. 2.3. Statistical analysis Associations between different underlying conditions and hospitalisation with faecal carriage of ESBL-PE were analysed using x2 and Fisher’s exact tests. Pair-wise analysis was conducted using IBM SPSS Statistics for Windows v.20.0 (IBM Corp., Armonk, NY). A P-value of <0.05 was considered statistically significant in all cases. 2.4. Ethical clearance This study was approved by the Institutional Ethics Committee of Amrita Institute of Medical Sciences. Informed consent was obtained from all of the participants before enrolment. 3. Results Among the 480 individuals screened for ESBL-PE, 260 were asymptomatic healthy individuals who came for health check-up and 220 were hospitalised patients admitted for >72 h in the hospital. Both groups had a preponderance of males [69.6% (181/260) among healthy individuals and 61.4% (135/220) among hospitalised patients]. The mean patient age was 45.5 years and 52.4 years for healthy individuals and hospitalised patients, respectively. Of the 220 hospitalised patients screened, the various underlying conditions (patients could have more than one underlying condition) were diabetes mellitus (63; 28.6%), coronary artery disease (23; 10.5%), malignancy (46; 20.9%), gastrointestinal
151
Table 1 Faecal carriage rate of extended-spectrum b-lactamase-producing Enterobacteriaceae among hospitalised patients (n = 220) and healthy individuals (n = 260). Hospitalised patients [n (%)]
Strain Escherichia coli Klebsiella pneumoniae Others Total
Healthy individuals [n (%)]
97 (44.1) 37 (16.8) 4 (1.8)
69 (26.5) 15 (5.8) 4 (1.5)
138 (62.7)
88 (33.8)
disorders (99; 45.0%), transplantation (3; 1.4%) and renal disease (26; 11.8). Statistical analysis of hospitalised patients with respect to different underlying diseases for faecal carriage of ESBL-PE showed none of them to be significant. However, when hospitalised patients were compared with healthy individuals, the former had a significant association (P < 0.001). All hospitalised patients had varying degrees of exposure to broad-spectrum antibiotics such as third-generation cephalosporins, fluoroquinolones, aminoglycosides and carbapenems. The mean duration of hospital stay in these patients was 16.23 days, and of these 30 patients died during the course of their treatment. The overall faecal carriage rate of ESBL-PE was 62.7% (138/220) among hospitalised patients and 33.8% (88/260) in healthy asymptomatic individuals. The different ESBL-PE species isolated are shown in Table 1. Antibiotic susceptibility testing of 51 representative isolates from healthy individuals and 61 isolates from hospitalised patients revealed a high degree of resistance in the latter. Cross-resistance to fluoroquinolones was the highest in both groups. Genotypic characterisation of representative ESBL-PE isolates (51 from healthy individuals and 53 from hospitalised patients) revealed a predominance of CTX-M genes (77–88%) (Table 2). The various combinations in which the ESBL genes (TEM, SHV and CTXM) were present in these isolates are given in Table 2. The presence of all the three ESBL genes was more often seen in Klebsiella spp. compared with E. coli. The presence of CTX-M and its association with cross-resistance to other antibiotics was analysed. Resistance to ciprofloxacin was the highest, with 81% in isolates from hospitalised patients and 60% in isolates from healthy individuals. The other notable feature was the emergence of carbapenem resistance (14.8%; 9/61) in ESBL-PE isolates from hospitalised patients and their absence in isolates from healthy individuals. The actual carbapenem resistance rate in hospitalised patients is expected to be ca. 20–30% considering the fact that only 61 isolates were eventually tested. Carbapenemresistant isolates were predominantly K. pneumoniae (5/9), followed by E. coli (2/9) and Enterobacter spp. (2/9). The majority (6/9) of these patients were also being treated with carbapenems.
Table 2 Genotypic distribution of extended-spectrum b-lactamase (ESBL) genes in ESBLproducing Enterobacteriaceae isolates from hospitalised patients and healthy individuals (n = 53/51). ESBL gene
Escherichia Klebsiella Enterobacter coli (n = 34/37) pneumoniae (n = 17/13) spp. (n = 2/1)
CTX-M SHV TEM CTX-M and SHV SHV and TEM CTX-M and TEM CTX-M, SHV and TEM None
16/26 1/0 3/0
9/5 2/0 3/4
3/4
4/2 1/1 1/2 7/2 1/1
1/0 1/0
152
R. Babu et al. / Journal of Global Antimicrobial Resistance 6 (2016) 150–153
Table 3 Comparison of faecal carriage rates of extended-spectrum b-lactamase-producing Enterobacteriaceae (ESBL-PE) from different studies in India. Author/year
Location
Deshpande et al., 2012 [7] Rodrigues et al., 2005 [8] Mathai et al., 2015 (n = 22) [5] Vandana et al., 2010 [9] Kothari et al., 2013 (n = 27) [10] Sarma and Ahmed, 2010 [11] Azim et al., 2010 [12] Present study, 2013 (n = 106)
Mumbai Mumbai Vellore Manipal New Delhi Guwahati Lucknow Kochi
Faecal carriage rate of ESBL-PE (%)
ESBL gene rate (%)
Hospitalised patients
Healthy individuals
CTX-M
SHV
TEM
23.9 11 19
ND ND 95.5 ND 82.5 ND ND 83
ND ND 9 ND ND ND ND 20
ND ND 68 ND ND ND ND 34
18.5 20.6 26 92 62.7
33.8
ND, not done.
4. Discussion Faecal carriage of ESBL-PE is considered to be a risk factor for infection with these bacteria. Previous studies have documented high rates of infections such as urinary tract infection due to ESBLPE in patients who are also intestinal carriers of these pathogens [3]. Faecal carriage rates also vary with geographical location owing to factors such as hygiene, sanitation, availability of antibiotics etc. playing a major role in the dissemination of these bacteria. Previous Indian studies have reported faecal carriage rates ranging from 18% to 92% among hospitalised patients and from 11% to 24% among healthy individuals (Table 3) [5,7–12]. The present study reports a very high rate (62.7%) of faecal carriage among hospitalised patients, but this is still less than the 92% reported by Azim et al. from Lucknow, India [12]. The high rates can be explained by the fact that most of the patients had chronic illness and had undergone multiple admissions and courses of antibiotics. Among the healthy individuals, the faecal carriage rate of ESBL-PE was 33.8%, which is one of the highest reported in the literature. An identical study by Rodrigues et al. a decade earlier among healthy executives in Mumbai showed a rate of 11% [8]. One reason for such high carrier rates could be the urban nature of the population where prior use of antibiotics in the last 3 years cannot be discounted. Moreover, prior exposure to thirdgeneration cephalosporins and fluoroquinolones is not a prerequisite for acquiring ESBL-PE, as Mathai et al. reported a faecal carriage rate of 19% among an antibiotic-naı¨ve healthy tribal population with no access to healthcare facilities [5]. In 2012, Luvsansharav et al. reported an alarmingly high rate of 65.7% of CTX-M-positive ESBL-PE in a rural Thai community [13]. Close contact with colonisers and patients coupled with exposure to common sources such as contaminated food and water and poor sanitation have been suggested for their spread in the community [14]. The emergence of carbapenem-resistant Enterobacteriaceae isolates (14.8%) among hospitalised patients was also expected due to the widespread use of carbapenems for treating ESBL-associated infections. A study from Pakistan has already reported 18.5% faecal carriage of NDM-1 carbapenemase-producing isolates among hospitalised patients [15]. A recent report from Bangladesh also reported NDM-1 faecal carriage in patients with diarrhoea [16]. The total absence of carbapenem resistance among healthy individuals is reassuring, but with reports of isolation of carbapenem-resistant isolates from the environment it is only a matter of time before we start encountering intestinal colonisation by these isolates [17]. The predominance of CTX-M ESBL genes is also along the expected lines, as previous studies have reported CTX-M-15 to be the most common ESBL gene among Indian isolates (Table 3) [5,18,19]. The high degree of cross-resistance to fluoroquinolones among CTX-M-positive ESBL-PE can be explained by the cotransfer of plasmid-mediated qnr genes [18].
Therefore, it is not surprising that the first report describing CTX-M-15 and co-transfer of the qnrB plasmid-mediated gene along with CTX-M-15 were from Indian isolates [18]. CTX-M alleles also accounted for >90% of ESBLs among community ESBL-PE worldwide [1]. Historically, faecal carriage rates in Europe have rarely exceeded 10% in the community; in contrast, Thailand has reported rates as high as 69% [13]. Many European studies have documented high rates of acquisition of ESBL-PE associated with travel to Southeast Asia, Pakistan and the Middle East [2]. Acquisition rates as high as 73% have been reported in German travellers returning from India [2]. Studies on the clonality of ESBLproducing E. coli have shown the presence of diverse clones in the community. However, the presence of ST131 clone in India, Pakistan and Madagascar has been reported previously [20]. The dramatic increase in faecal carriage rates of ESBL-PE worldwide has been attributed to the efficient transfer of the plasmid-mediated blaESBL gene between Gram-negative bacteria. In a background of poor sanitation, ESBL-producing faecal coliforms (E. coli) can spread like wild fire leading to asymptomatic intestinal colonisation of the whole community, which then acts as a reservoir and subsequently leading to community-acquired infections in susceptible individuals. Despite the growing evidence on colonisers acting as a reservoir for nosocomial infections, routine screening of patients before admission is not recommended. Standard contact precautions and good infection control practices are all that is recommended for such patients. 5. Conclusion The faecal carriage rate of ESBL-PE among hospitalised patients was almost double that of healthy individuals. Carriage of carbapenem-resistant isolates is emerging among hospitalised patients and is a threat to the future of antibiotic chemotherapy. The high burden of ESBL-PE among healthy asymptomatic individuals has significant public health implications, including the treatment of community-acquired infections. The developing world, especially India, is staring at a pandemic of ESBL-PE. CTX-Mproducing E. coli are true community ESBL and their dissemination into the water supply and food chain needs to be prevented. The spread of these organisms in the community merits radical measures to improve sanitation and implement antibiotic stewardship. The major limitations of this study were that susceptibility testing, clonal typing for ST131 and genotyping for all ESBLproducing and carbapenem-resistant isolates were not performed. In the absence of sequencing, mere amplification of SHV and TEM genes may not necessarily mean ESBL production. Funding None.
R. Babu et al. / Journal of Global Antimicrobial Resistance 6 (2016) 150–153
Competing interests None declared. Ethical approval This study was approved by the Institutional Ethics Committee of Amrita Institute of Medical Sciences (Kochi, India). Informed consent was obtained from all of the participants before enrolment.
[10]
[11]
[12]
[13]
References [1] Woerther PL, Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extended-spectrum b-lactamases in the community: toward the globalization of CTX-M. Clin Microbiol Rev 2013;26:744–58. [2] Lu¨bbert C, Straube L, Stein C, Makarewicz O, Schubert S, Mo¨ssner J, et al. Colonization with extended-spectrum b-lactamase-producing and carbapenemase-producing Enterobacteriaceae in international travelers returning to Germany. Int J Med Microbiol 2015;305:148–56. [3] Tan BH, Tan AL. Community-acquired bacteremia with an ESBL-producing Escherichia coli strain—a case report. Int J Infect Dis 2002;6:318–9. [4] Arpin C, Dubois V, Maugein J, Jullin J, Dutilh B, Brochet JP, et al. Clinical and molecular analysis of extended-spectrum b-lactamase-producing enterobacteria in the community setting. J Clin Microbiol 2005;43:5048–54. [5] Mathai D, Kumar VA, Paul B, Sugumar M, John KR, Manoharan A, et al. Fecal carriage rates of extended-spectrum b-lactamase-producing Escherichia coli among antibiotic naive healthy human volunteers. Microb Drug Resist 2015;21:59–64. [6] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; approved standard—ninth edition. Document M2-A9. Wayne, PA: CLSI; 2013. [7] Deshpande P, Vadwai V, Shetty A, Dalal R, Soman R, Rodrigues C. No NDM-1 carriage in healthy persons from Mumbai: reassuring for now. J Antimicrob Chemother 2012;67:1046–7. [8] Rodrigues C, Shukla U, Jog S, Mehta A. Extended-spectrum b-lactamaseproducing flora in healthy persons. Emerg Infect Dis 2005;11:981–2. [9] Vandana KE, Varghese G, Krishna S, Mukhopadhyay C, Kamath A, Ajith V. Screening at admission for carrier prevalence of multidrug-resistant organisms
[14]
[15]
[16]
[17]
[18]
[19]
[20]
153
in resource-constrained settings: a hospital-based observational study. J Hosp Infect 2010;76:180–1. Kothari C, Gaind R, Singh LC, Sinha A, Kumari V, Arya S, et al. Community acquisition of b-lactamase producing Enterobacteriaceae in neonatal gut. BMC Microbiol 2013;13:136. Sarma JB, Ahmed GU. Prevalence and risk factors for colonisation with extended spectrum b-lactamase producing Enterobacteriaceae vis-a`-vis usage of antimicrobials. Indian J Med Microbiol 2010;28:217–20. Azim A, Dwivedi M, Rao PB, Baronia AK, Singh RK, Prasad KN, et al. Epidemiology of bacterial colonization at intensive care unit admission with emphasis on extended-spectrum b-lactamase- and metallo-b-lactamase-producing Gram-negative bacteria—an Indian experience. J Med Microbiol 2010;59: 955–60. Luvsansharav UO, Hirai I, Nakata A, Imura K, Yamauchi K, Niki M, et al. Prevalence of and risk factors associated with faecal carriage of CTX-M b-lactamase-producing Enterobacteriaceae in rural Thai communities. J Antimicrob Chemother 2012;67:1769–74. Valverde A, Grill F, Coque TM, Pintado V, Baquero F, Canto´n R, et al. High rate of intestinal colonization with extended-spectrum-b-lactamase-producing organisms in household contacts of infected community patients. J Clin Microbiol 2008;46:2796–9. Day KM, Ali S, Mirza IA, Sidjabat HE, Silvey A, Lanyon CV, et al. Prevalence and molecular characterization of Enterobacteriaceae producing NDM-1 carbapenemase at a military hospital in Pakistan and evaluation of two chromogenic media. Diagn Microbiol Infect Dis 2013;75:187–91. Islam MA, Nabi A, Rahman M, Islam M, Ahmed D, Faruque AS, et al. Prevalence of faecal carriage of NDM-1-producing bacteria among patients with diarrhoea in Bangladesh. J Med Microbiol 2014;63:620–2. Walsh TR, Weeks J, Livermore DM, Toleman MA. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 2011;11: 355–62. Jacoby GA, Walsh KE, Mills DM, Walker VJ, Oh H, Robicsek A, et al. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother 2006;50:1178–82. Muzaheed Y, Doi Y, Adams-Haduch JM, Shivannavar CT, Paterson DL, Gaddad SM. Faecal carriage of CTX-M-15-producing Klebsiella pneumoniae in patients with acute gastroenteritis. Indian J Med Res 2009;129:599–602. Herindrainy P, Randrianirina F, Ratovoson R, Ratsima Hariniana E, Buisson Y, Genel N, et al. Rectal carriage of extended-spectrum b-lactamase-producing Gram-negative bacilli in community settings in Madagascar. PLoS ONE 2011;6:e22738.