Surveillance of Phenotypic Extended Spectrum Beta-Lactamase Resistance in Blood Isolates at a Hospital in East Trinidad

Rajeev P Nagassar¹, Keston Daniel¹, Roma J Bridgelal-Nagassar², Khalil Ashraph³

¹The Eastern Regional Health Authority
²The North West Regional Health Authority
³Department of Paraclinical Sciences, The University of the West Indies, Trinidad and Tobago

Corresponding Author

Rajeev P Nagassar
The Eastern Regional Health Authority
Trinidad and Tobago
Email: [email protected]

DOI: 10.48107/CMJ2021.04.008

Copyright: This is an open-access article under the terms of the Creative Commons Attribution License which permits use, distribution, and reproduction in any medium, provided the original work is properly cited.

©2021 The Authors. Caribbean Medical Journal published by Trinidad & Tobago Medical Association

ABSTRACT

Background

The Centres for Disease Control and Prevention (CDC) and World Health Organization (WHO) list extended spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae as serious threats and priority pathogens. This study identified phenotypic resistance patterns to these pathogens in east Trinidad, West Indies. We also aimed to set up and test a pilot surveillance system aligned to WHO’s Global Antimicrobial Surveillance System (WHO-GLASS).

Methods

Two key bacterial isolates, Escherichia coli and Klebsiella pneumoniae were used and one specimen, blood, was used to test a pilot surveillance system. Data for resistance patterns, for Sangre Grande Hospital (SGH), for ESBL producing E. coli and K. pneumoniae were downloaded from the Microscan Autoscan^© for the period 2013 – 2016. ESBL presence in bacteria resistant to Cefotaxime (CTX), Ceftazidime (CAZ) and Ceftriaxone (CRO) were recorded. Data were stored in a Microsoft Excel^© spreadsheet and inputted into IBM^© SPSSv22. Data were displayed as resistance percentages for the year. No patient data were collected. Simple descriptive statistics were used.

Results

The number of organisms recovered from the database for the period 2013 to 2016 were:134E. coli and 59K. pneumoniae. Phenotypic resistance rates for ESBLs for 2013 to 2016 were:

E. coli:

2013: Resistance ranged from 22.2-29.6% with maximum resistance seen for CTX.
2014: Resistance ranged from 12.9- 22.2%, with maximum resistance seen for CRO.
2015: Resistance ranged from 21.4- =26.2%, with maximum resistance seen for CTX.
2016: Resistance ranged from 29.4- 32.4%, with maximum resistance seen for CRO and CTX.

K. pneumoniae:

2013: Resistance was 40% for all 3^rd generation Cephalosporins.
2014: Resistance was 16.7% for all 3^rd generation Cephalosporins.
2015: Resistance was 16.7% for all 3^rd generation Cephalosporins.
2016: Resistance ranged from 52.6 – 63.2%, with maximum resistance seen for CAZ.

Conclusion

Phenotypic resistance rates in K. pneumoniae and E. coli were generally high. There was an overall increase in resistance from 2013 to 2016 for both K. pneumoniae and E. coli with greater resistance being seen in K. pneumoniae.

Key Words: ESBL, Escherichia coli, Klebsiella pneumoniae, Resistance

INTRODUCTION

The World Health Organization (WHO) has developed a global priority pathogens list (global PPL) of antibiotic resistant bacteria to assist in prioritising the research and development of new and effective antibiotic treatments.¹ This includes the priority 1 pathogen Enterobacteriaceae, including phenotypic carbapenem resistant and third generation cephalosporin-resistant strains. The Centres for Disease Control and Prevention (CDC) lists extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae as serious threats.² Hence, we chose to study ESBL production in these two bacteria. This data formed the rationale for choosing to study ESBL production in these two bacteria.

One study from Nepal indicated a high rate of extended spectrum beta-lactamase (ESBL) production was found in the E. coli and K. pneumoniae isolated from outpatients. This study suggested dissemination of ESBL producing bacteria in the community.³ Chong et al have indicated that there is worldwide spread of ESBL producing bacteria which is of critical concern.⁴ Heinz et al have found resistance genes circulating in the Caribbean region.⁵ In a study from Jamaica by Nicholson et al, it was found that 18.2% of K. pneumoniae produced ESBLs, while there were no ESBL producing E. Coli.⁶ This resistance in K. pneumoniae was also found by Christian et al in Jamaica.⁷ In Trinidad and Tobago, ESBL producing K. pneumoniae have also been found.⁸ In fact, Akpaka et al found ESBLs in E. coli and K. pneumoniae doing phenotypic studies using the Microscan automated system compared to the E-test.⁹ Surveillance of antibiotic resistance is important for policy, infection prevention and control and antibiotic stewardship. This surveillance must first have a testing system at a designated laboratory and then a method of generation of key indicator data. This is seen in the WHO Global Antimicrobial Surveillance System (GLASS) methodology.¹⁰

In this paper, we examine ESBL resistance in E. coli and K. pneumoniae in blood isolates at the Sangre Grande Hospital. The aim was to initiate an antimicrobial resistance surveillance system, starting with these key bacterial isolates, which can be expanded to include other pathogens and resistance mechanisms in the future.

METHODS

The site of the study was the Sangre Grande Hospital (SGH) which has 120 beds and serves a population of 120,000 persons. This is the smallest population served by a hospital in Trinidad and Tobago.¹¹ This study was a descriptive surveillance study utilising retrospective data. The objective was to use one key, but clinically important specimen. Thus, blood was chosen. In addition, two key Gram-negative organisms were tested against third generation cephalosporins and using the third generation cephalosporin plus a beta-lactamase inhibitor as outlined below. This study was aligned to the WHO-GLASS methods and recommendations and methods previously published by Akpaka et al in 2008. Data for resistance patterns for SGH for ESBL producing Escherichia coli and Klebsiella pneumoniae were downloaded from the Microscan Autoscan^© for the period 2013 to 2016. This was for blood isolates only, in line with WHO-GLASS methods. This data represented isolates from the community and hospital. The study utilised isolates only and no demographic or patient data were collected. Simple descriptive statistics were used.

Quality Control

The integrated LabPro^© 2.0 version from the Microscan included the Alert expert system. This system used growth in the presence of ceftazidime and cefotaxime compared with cefotaxime/clavulanate and ceftazidime/clavulanate. This comparison is used in the Gram-negative panel for ESBL screening. ATCC 25922, an E. coli, was used for quality control during the years 2013 to 2016. The data was inspected in a Microsoft Excel^© spreadsheet to ensure that the data quality was met. Duplicate isolates were removed to ensure that there were no repeat samples from the same patient. The LabPro generates reports according to international standard criteria. The software was last updated in 2016. Once an ESBL was detected in LabPro, all the third generation cephalosporins were flagged as resistant.

Data Management

Extended Spectrum Beta-Lactamase (ESBL) production was screened by the Microscan. It measured changes in the microtiter wells. All positive ESBL producers were recorded. Once an ESBL was detected, cefotaxime (CTX), ceftazidime (CAZ) and ceftriaxone (CRO) were flagged as resistant by the Microscan’s software. This study showed almost 100% concordance between the E-test and Microscan. Data were stored in a Microsoft Excel^© spreadsheet and inputted into IBM^© SPSSv22. Data were displayed as resistance percentages for the year. WHONET software was also used for data analysis of resistance patterns. Clinical Laboratory Standards Institute (CLSI) interpretative criteria were used for comparison. The period 2013 to 2016 was examined to determine if there was any change in resistance patterns of ESBLs.

RESULTS

The number of organisms recovered from the database for the period 2013 to 2016, was 134 E. coli and 59 K. pneumoniae as distributed in Table 1. For the year 2013, 27 E. coli and 10 K. pneumoniae were isolated. For the years 2014, 2015 and 2016, 31 E. coli and 12 K. pneumoniae, 42 E. coli and 18 K. pneumoniae and 34 E. coli and 19 K. pneumoniae were isolated, respectively (see Table 1). Phenotypic resistance rates for ESBLs for 2013 to 2016 were:

Table 1: Number of Organisms for the period 2013 to 2016

E.coli:
The resistance, as indicated by phenotypic ESBL presence, for cefotaxime (CTX), ceftazidime (CAZ) and ceftriaxone (CRO) fluctuated from 2013 to 2016. There, however, was a small increase from 22.2%-29.6% in 2013 to 29.4%- 32.4%, in 2016. This is highlighted in Table 2 and Figure 1.

Figure 1: Percent ESBL Resistance for Escherichia coli isolated from blood in Sangre Grande Hospital 2013 – 2016

Figure 2: Percent ESBL Resistance for Klebsiella pneumoniae isolated from blood in Sangre Grande Hospital 2013 – 2016

Table 2: Phenotypic ESBL Positive Escherichia coli isolated from blood in Sangre Grande Hospital 2013 – 2016

K pneumoniae:
The resistance, as indicated by phenotypic ESBL presence, for cefotaxime (CTX), ceftazidime (CAZ) and ceftriaxone (CRO) fluctuated from 2013 to 2016. It was, however, quite high overall and showed a trend of increased resistance from 40% in 2013 to 63.2% in 2016. This is highlighted in Table 3 and Figure 2.

Table 3: Phenotypic ESBL Positive Klebsiella pneumoniae isolated from blood in Sangre Grande Hospital 2013 – 2016

DISCUSSION
E. coli resistance to the third generation cephalosporins tested and thus ESBLs was over 30% by 2016 indicating a trend of a high level of resistance. Similarly, K. pneumoniae, also showed increasing ESBL detection from 2013 to 2016. This is consistent with K. pneumoniae resistance in Trinidad and elsewhere in the Caribbean.^8-9 In a phenotypic study from 2004 to 2007, Akpaka et al showed that the ESBL rate among the K. pneumoniae isolates was 15.2% and 9.3% among the E. coli isolates.⁹

Secondly, in a study carried out by Cherian et al, the authors also found resistance in K. pneumoniae and E. coli, but at rates lower than the study by Akpaka et al and the current study.^{9, 12} Thus, the rates of phenotypic ESBL production were much higher in our setting for both organisms. The increasing resistance from the study by Cherian et al in 1998 and Akpaka et al to the current study period indicates that there is a temporal trend where resistance increases with time.⁹ The other notable fact is that with all studies done in Trinidad and Tobago, the K. pneumoniae shows greater resistance over the periods studied compared to E. Coli.^{9, 12} This is an essential epidemiological trend. Importantly, the study by Akpaka et al was conducted over a similar time to this current study, but involved a much larger hospital serving a larger population and thus, they would have expectedly retrieved more isolate information. Our study was therefore limited by the smaller population served.

Additionally, K. pneumoniae and E. coli resistance are both quite concerning globally. The Global Antimicrobial Resistance Surveillance System (GLASS) Report from 2017 – 2018 shows high rates of resistance in K. pneumoniae and E. coli globally.¹³ In fact, Trinidad and Tobago is now contributing to this data as the country has enrolled in 2020 (verbal communication). A study from Taiwan showed that 19.7% of patients with community onset bacteraemia had third generation cephalosporin resistance signifying that ESBL resistance in E. coli and K. pneumoniae is not just a local problem, but a regional and international issue requiring robust surveillance and testing systems which are simple to implement, perform and sustain.¹⁴

Importantly, an ESBL is an enzyme produced by certain Enterobacteriae (E. coli, Klebsiella pneumoniae) enabling them to hydrolyse all penicillins, aztreonam, cephalosporins, but not cephamycins like (cefoxitin, cefotetan) or carbapenems.¹⁵ This study looked at screening methods for ESBLs and its use for surveillance. Confirmatory methods include the double disc diffusion, combination disc test, E-test, and molecular methods.^16-17 The Microscan did use ceftazidime with clavulanic acid and cefotaxime with clavulanic acid for screening.¹⁸ When reporting results, it should indicate resistance to all penicillins, all cephalosporins and aztreonam, even if they are susceptible in vitro. In confirmed or screening positive ESBL producing bacteria, reports should have no change in susceptibility interpretations for cephamycins (e.g., cefoxitin), beta-lactam/beta-lactamase inhibitor combinations (e.g., piperacillin/ tazobactam) and carbapenems.¹⁹ However, it may not be practical to use beta-lactam/beta-lactamase inhibitor combinations in vivo as they may be ineffective.²⁰

Lastly, this study was limited because there were no confirmatory ESBL tests which can be either phenotypic or molecular based. This may have resulted in an overestimation of resistance. However, the study by Akpaka et al shows that there was an almost 100% concordance between the confirmatory test, the E-test, and the Microscan results. Thus, there has been previous validation by Akpaka et al, even though it was not stated, of Microscan results for ESBL testing in surveillance organisms used in our study.⁹ This study also did not look at diseases such as septicaemia as it was a laboratory-based study and only dealt with laboratory isolate data which consisted of blood isolates and no other samples or clinical information. Thus, resistance from other sample types was not considered. This would have led to a lower level of resistance reported overall and lower number of isolates recovered for the time period. Funding was also limited and thus, we were unable to procure reagents and material for confirmatory testing. There were a relatively small number of organisms retrieved from the database but the hospital was also small serving a small population.

CONCLUSION
In conclusion, this study conducted 10 years after the last phenotypic report by Akpaka et al, published in 2008, shows that phenotypic ESBL resistance is still a problem, at least in east Trinidad. Thus, clinicians should be mindful of this in assessing microbiology reports and treating patients. Most of the regular work done in laboratories is phenotypic. This sets out a practical approach to finding ESBL resistance for under-resourced laboratories. We believe that this pilot surveillance study, supported by the previous validation of the Microscan results, will contribute to the knowledge needed to combat the evolving global problem of antimicrobial resistance and will help inform rational antibiotic treatment. This is important for policy and guideline development in Antimicrobial Resistance (AMR) in Trinidad and Tobago, the Caribbean region and globally.

Ethical Approval Statement: The Ethics Committee of the Eastern Regional Health Authority approved this study.

Conflict of Interest: None

Informed Consent Statement: Not applicable

Funding Statement: None

Author Contributions: Rajeev P. Nagassar, Roma J. Bridgelal-Nagassar, Keston Daniel and Khalil Ashraph designed, analysed and wrote the final paper. Rajeev P. Nagassar, Roma J. Bridgelal-Nagassar, Keston Daniel and Khalil Ashraph approved the final paper. Keston Daniel and Khalil Ashraph analysed the data and reviewed the final paper for publication.

REFERENCES

1. World Health Organization. Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of new Antibiotics. https://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf?ua=1 (Accessed 10/01/2019).
2. Centres for Disease Control and Prevention. Antibiotic / Antimicrobial Resistance (AR / AMR). 2020. https://www.cdc.gov/drugresistance/biggest-threats.html#extend (Accessed30/08/ 2020).
3. Nepal K, Pant ND, Neupane B, Belbase A, Baidhya R, Shrestha RK et al. Extended spectrum beta-lactamase and metallo beta-lactamase production among Escherichia coli and Klebsiella pneumoniae isolated from different clinical samples in a tertiary care hospital in Kathmandu, Nepal. Ann Clin Microbiol Antimicrob. 2017; 16(1):62.
4. Chong Y, Shimoda S, Shimono N et al. Current epidemiology, genetic evolution and clinical impact of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae. Infect Genet Evol. 2018; 61:185-188.
5. Heinz E, Brindle R, Morgan-McCalla A, Peters K, Thomson NR. Caribbean multi-centre study of Klebsiella pneumoniae: whole-genome sequencing, antimicrobial resistance and virulence factors. MicrobGenom. 2019;5(5): e000266. doi:10.1099/mgen.0.000266.
6. Nicholson AM, Gayle P, Roye-Green K. Extended spectrum beta-lactamase producing organisms at the University Hospital of the West Indies. WIMJ. 2004; 53(2):104-8.
7. Christian NA, Roye-Green K, Smikle M. Molecular epidemiology of multidrug resistant extended spectrum beta-lactamase producing Klebsiella pneumoniae at a Jamaican hospital, 2000-2004. BMC Microbiol. 2010; 10:27.
8. Cheddie P, Dziva F, Akpaka, PE. Detection of a CTX-M group 2 beta-lactamase gene in a Klebsiella pneumoniae isolate from a tertiary care hospital, Trinidad and Tobago. Ann Clin Microbiol Antimicrob.2017; 16 (33). https://doi.org/10.1186/s12941-017-0209-x
9. Akpaka PE, Swanston WH. Phenotypic detection and occurrence of extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli at a tertiary Hospital in Trinidad & Tobago. Braz J Infect Dis. 2008; 12(6): 516-520. http://dx.doi.org/10.1590/S1413-86702008000600014.
10. World Health Organization. Global Antimicrobial Resistance Surveillance System: Manual for Early Implementation. 2015. https://apps.who.int/iris/bitstream/handle/10665/188783/9789241549400_eng.pdf?sequence=1 (Accessed 06/12/2020).
11. The Eastern Regional Health Authority. About Us. 2020. http://www.erha.co.tt/abouterha/#:~:text=Vision%20Statement,the%20needs%20of%20the%20population. (Accessed 06//12/2020).
12. Cherian B.P., Manjunath M., Pereira L.M., Prabhakar P. Extended spectrum β-lactamase producing Enterobacteriaceae in a tertiary care hospital in Trinidad & Tobago. West Indian Med J.2000; 52:31-3.
13. World Health Organization. Global Antimicrobial Resistance Surveillance System (GLASS) Report Early implementation. 2018. https://apps.who.int/iris/bitstream/handle/10665/279656/9789241515061-eng.pdf?ua=1 (Accessed 30/08/2020).
14. Lin WP, Huang YS, Wang JT, Chen YC, Chang SC. Prevalence of and risk factor for community-onset third-generation cephalosporin-resistant Escherichia coli bacteraemia at a medical centre in Taiwan. BMC Infect Dis. 2019;19(1):245. doi:10.1186/s12879-019-3880-z
15. Centres for Disease Control and Prevention. ESBL-producing Enterobacteriaceae in Healthcare Settings. 2019. https://www.cdc.gov/hai/organisms/ESBL.html (Accessed 31/08/2020).
16. Poulou G, Grivakou E, Vrioni G et al. Modified CLSI Extended-Spectrum β-Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases. J Clin Microbiol. 2014; 52 (5): 1483-1489. DOI: 10.1128/JCM.03361-13
17. Drieux L, Brossier F, Sougakoff W, Jarlier V. Phenotypic detection of extended-spectrum beta-lactamase production in Enterobacteriaceae: review and bench guide [published correction appears in Clin Microbiol Infect. 2008 May;14 Suppl 5:21-4]. Clin Microbiol Infect. 2008;14 Suppl 1:90-103. doi:10.1111/j.1469-0691.2007. 01846.x
18. Winstanley T, Courvalin P. Expert Systems in Clinical Microbiology. Clin Microbiol Rev. 2011; 24 (3): 515-556. DOI: 10.1128/CMR.00061-10
19. Lalitha MK. Manual on Antimicrobial Susceptibility Testing. ijmm.org%2Fdocuments%2FAntimicrobial.doc (Accessed 31/08/2020).
20. Harris PNA, Tambyah PA, Lye DC, et al. Effect of Piperacillin-Tazobactam vs Meropenem on 30-Day Mortality for Patients with E coli or Klebsiella pneumoniae Bloodstream Infection and Ceftriaxone Resistance: A Randomized Clinical Trial [published correction appears in JAMA. 2019 Jun 18;321(23):2370]. JAMA. 2018;320(10):984-994. doi:10.1001/jama.2018.12163