Prevalence of extended-spectrum beta-lactamase producing bacteria from animal origin: A systematic review and meta-analysis report from India

Antimicrobial resistance (AMR) due to the emergence and spread of extended-spectrum beta-lactamase (ESBL) producing bacteria are becoming a serious global public health concern. This article aims to assess the overall prevalence of ESBLs among animals in India, with year-wise, zone-wise and species-wise stratification. Systematic search from PubMed, Google Scholar and J-Gate Plus was carried out and 24 eligible articles from 2013–2019 in India were retrieved. The R Open source Scripting software was used to perform statistical analysis. The overall prevalence of ESBLs among animals in India was 9%. The pooled prevalence of ESBLs in animals were 26, 11, 6 and 8% for north, east, south and central zones, respectively. The reported prevalence of ESBLs in animals were 12, 5, 8, 8, 12, 13 and 33% were reported for the years 2013, 2014, 2015, 2016, 2017, 2018, 2019 respectively. The species-wise stratified results showed a predominance of ESBL producing Klebsiella pneumoniae strains (11%) when compared to Escherichia coli and Pseudomonas spp. which were 7% and 5%, respectively. The prevalence data generated could be utilized in infection control and in antibiotic use management decisions for developing appropriate intervention strategies.


Introduction
Antimicrobial resistance (AMR) has been universally recognized as an emerging global problem to public health. Although the prevalence of AMR is sporadic, it is widespread in the Asian region. India, located in the southern part of Asia, marks a high, immeasurable burden of AMR among livestock due to poor documentation, sub-standard regulations with a shortfall in forbidding protocol enforcement [1]. This study aims to estimate the pooled prevalence of Extended-spectrum β-lactamases (ESBLs) in India by conducting systematic review and metaanalysis with 23  antimicrobial agents are the most favored class of antimicrobials for the treatment of bacterial infections, hence becoming the main cause of resistance to β-lactam antibiotics, globally [2]. Prevalence of ESBLs producing Klebsiella is becoming a major concern in China, Korea, Japan and India [3]. ESBLs enzymes are produced by the gram-negative bacteria to incur resistance against the β-lactams. Klebsiella pneumoniae and Escherichia coli are the main gram-negative bacteria producing ESBLs [4]. However Proteus mirabilis, Enterobacter spp., Salmonella, Acinetobacter baumannii, and Pseudomonas aeruginosa also produce ESBLs to acquire resistance [5].
The incessant liability of gram-negative strains to a myriad β-lactams has begotten rapid and vigorous production and mutation of β-lactamases in these bacteria, hence, incurring resistance against the newly developed β-lactam antibiotics [2]. Treatment for these disease causing multidrug-resistant (MDR) organisms is a therapeutic challenge. The risk factors for developing infection with ESBL-producing organisms include indiscriminate and off-label use of antibiotics [6]. At present, animals without any recognized risk factor for multidrug-resistant organisms are found to have ESBL-producing organisms. Hence, diagnosis of ESBL-producing organisms has become vital [7]. MDRs are posing a treatment challenge, and a major cause of morbidity and mortality worldwide [1]. Unfortunately, India, being a developing country, does not have an adequate surveillance system that could track indiscriminate use or consumption of antibiotics in livestock populations. This meta-analysis will improve our understanding of the distribution of ESBLs in India. A set of similar events for which a study is conducted is called a population, in our study it refers to poultry, bovine and birds. The outcome of our study would indicate the prevalence of ESBLs by zone, year and species in India. It is a quantitative, epidemiological study designed to systematically assess the previous research studies to derive the conclusions of this research [8]. This study highlighted the prevalence of ESBL from the time period 2013-2019, with zone-wise and species-wise prevalence of ESBLs in India. A priori protocol was followed for this study with reference to a work done by Bulabula and co-workers [9]. To our knowledge, this is the first meta-analysis report from India on animals, which would aid in updating the national treatment guidelines for ESBL infections among animals.

Literature search
A Systematic search was conducted in "Pub Med", "Google Scholar" and "J-Gate-Plus" databases from Jan 2013 to May 2019 using the search terms "ESBL", "prevalence", "India", "Animals", "Poultry", "Cattle" and "Bovine" in combinations. Bibliographies of eligible studies were also manually searched to identify additional significant articles. A comprehensive search was conducted to ensure none of the research were missed out. The search was restricted to articles published in English.

Study selection criteria
All the articles that described the frequency of ESBL producing pathogens among the total isolates from animal samples (clinical/healthy) were considered eligible and included in the study. The qualified articles described the specific laboratory methods used to identify the ESBL producing pathogen along with species of the ESBL producing organism (Table 1). All the enrolled studies were restricted to India. Review articles, case reports and outbreaks were excluded.

Data extraction
For consistency, data was extracted independently by two people from selected articles. The data extracted from qualified studies included year of publication, first author, location where study was conducted, total sample size, strains detected ESBL positive, and method used for confirmation of ESBL producing pathogen. Any inconsistency in data collection was rectified by re-checking the articles for accuracy.

Quality assessment
Since it is a prevalence study, use of Newcastle-Ottawa scale is not recommend. However, quality assessment of the study was done on fixed rating scale. This scale includes evaluation of study selection, comparability and outcome, with each section having maximum number of stars as 5, 3 and 2 respectively. Hence, the overall quality assessment has a maximum score of 10 and minimum score for inclusion is 3 stars. Table 2 shows the risk of bias assessment for the studies included in quantitative synthesis.

Statistical analysis
Meta-analysis for the prevalence of ESBL producing pathogens among animal samples were conducted using the R Open source scripting software (version 3.4.3, R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/) [10]. The inbuilt packages used for analysis were Metafor and Meta R packages.
In the analysis, both random effect and fixed effect model were used to calculate the pooled prevalence of ESBL and I 2 statistic (to measure inconsistency). The τ 2 statistic was also calculated to measure the heterogeneity. Further, sub-group analysis was performed to reduce heterogeneity. In the present study, the data was stratified based on: year-wise (2013-2019) zonewise (North, East, West, South and Central zones) and species-wise (E. coli, Pseudomonas spp., and K. pneumoniae).

Distribution and characteristics of articles describing ESBLs in India
The electronic database searches returned 32 potential articles based on the keyword search. Review articles studying the ESBL prevalence in humans were excluded. A total of 23 articles were selected suitable for the study. The flowchart of systematic article selection is shown in Fig 1. All the articles included in the study described the prevalence of ESBL producing pathogens isolated from animals/animal samples from India. The maximum number of studies on this subject were found in the eastern zone followed by central, south and north zone. No studies were found from western zone of India. In total, 20 studies were on ESBLs produced by Escherichia coli, 6 on ESBLs produced by K. pneumoniae and 2 on ESBLs produced from Pseudomonas spp. The animal samples studied in the articles mainly included meat samples, milk samples, rectal swabs, cecal swabs and cloacal swabs from poultry birds, sheep, pig and cattle.

Discussion
Our study revealed that, the ESBL producing clinical isolates in India may not be very high, nonetheless it is significant. These drug-resistant pathogens are a serious concern worldwide  as they are associated with increase in morbidity and mortality rate due to infections they cause [31]. Extended-Spectrum Beta-Lactamases are produced by species of bacteria in order to inactivate antibiotics, causing antibiotic resistance. Beta-lactamase seems to be the prime cause in multidrug resistant (MDR) E. coli strains. Early detection of E. coli that produce beta lactamase is necessary in order to prevent MDR E. coli from spreading [32]. Activity of ESBLs caused by different beta-lactamases resulted in resistant genes within the farm [33]. The strains that were isolated showed that a small portion of the resistant genes were present in one farm [4] . The steep rise in income and the growing population has driven an increase in demand for animal products in India. India is one of the top consumers of antibiotics worldwide, it accounts for about 3% of global consumption which is estimated to double by 2030. This could be due to the non-therapeutic use of antibiotics in cases of prophylaxis and growth promotion [34]. Currently, the usage of antibiotics is high in poultry, swine and cattle production as compared to that being used by the human population [35][36].
To address the concern of antimicrobial resistant bacteria, it is crucial to raise awareness of the problem by collecting data on antibiotic resistance from various countries and regions. The paucity of studies available from India affirms attention for future research. To our  knowledge, this is the first meta-analysis regarding the magnitude of the ESBL problem in Indian animal population. From the 23 articles chosen in the study, the overall pooled prevalence of ESBL producing isolates from the animal samples was found to be 10%. In Asia, high rates of ESBL producing Enterobacteriaceae are seen with variation in the prevalence and the genotype of the ESBL producing isolates over the large geographical area [30].
The prevalence of ESBL producing isolates were 12,5,8,8,12,13 and 33% for the years 2013, 2014, 2015, 2016, 2017, 2018 and 2019 respectively, indicating an increase in the percent drug resistance since 2014 to 2019. The pooled prevalence of ESBL producing isolates was determined zone-wise and North zone showed a higher prevalence rate in comparison to other zones. Nonetheless, no studies on prevalence of ESBL producing isolates for animal samples from the Western zone of India are reported. Prevalence of species-wise classification was found to be 9, 10 and 5% for E. coli, K. pneumoniae and Pseudomonas spp. respectively, signifying that the ESBL producing K. pneumoniae is the most predominant ESBL producing isolate in India.
A study conducted in the intensive care units (ICUs) of an Indian hospital concluded that there is a need for constant surveillance to detect resistant bacterial strains, strict guidelines on antibiotic therapy, and effective infection control measures in order to reduce the spread of antibiotic resistant bacteria. The same study also revealed that there is a high number of ESBL producing E. coli in the ICUs of that hospital [31]. A study with pediatric and neonatal patients estimated the number of poor outcomes and indicated the association of blood stream infections (BSIs) with Extended-Spectrum Beta-Lactamase-producing Enterobacteriaceae (ESBL-PE). The results showed a high prevalence of BSIs due to ESBL-PE and increase in neonatal mortality [37][38][39]. A study from Germany demonstrated that direct transfer of ESBLproducing E. coli could occur between livestock and the farm workers who were in close contact with farm animals. The study also suggests an existence of epidemiological links between livestock and farm workers. A high prevalence of ESBL-producing E. coli in pig and cattle farms emphasizes the fact that livestock animals are a constant source for these potential human pathogens [33,[40][41]. Our research findings does have some minor limitations, which includes the lack of sufficient information on the prevalence of ESBL producers from different animal species. Upon advanced literature survey, we could find only a few articles that addressed the prevalence of ESBLs in animals.

Conclusion
India being a developing country, has the highest burden of bacterial infections. Hence, to combat this downfall, antibiotics are used widely and indiscriminately. The overuse, lack of awareness and non-therapeutic use of antibiotics is driving an increase in the antibiotic resistance among animals. This meta-analysis, indicated that the pooled prevalence of ESBLs for animals in India is not high, however, the overall prevalence remains significant at 10%. Additionally, only little information is currently available that addresses the prevalence of ESBLs in animals in India. The paucity of data on the clinical outcomes, magnitude and prevalence of the resistant ESBLs, calls for active surveillance which can help understand the epidemiology of ESBL burden in India. Furthermore, emphasis on awareness programs, personal and environmental hygiene should be implemented to stop and manage the spread of ESBLs to the animals and environment. Further studies are needed to better understand the complexity of the AMR problem in animal and human population.