Phylogenetically diverse Escherichia coli strains from chicken co-harbour multiple carbapenemase encoding genes (blaNDM-blaOXA-blaIMP)

Carbapenem resistant Enterobacteriaceae (CRE) has been public health risk in several countries and recent reports indicate the emergence of CRE in food animals. This study was conducted to investigate the occurrence, resistance patterns, and phylogenetic diversity of CRE E.coli from chicken. Routine bacteriology, PCR detection of E.coli species, multiplex PCR to detect carbapenemase encoding genes and phylogeny of CRE E. coli were conducted. The results show that 24.36 % (19/78) were identified as CRE based on the phenotypic identifications of which 17 were positive for the tested carabanemase genes. The majority, 57.99% (11/19) of the isolates harbored multiple carbapenemase genes. Four isolates harbored all blaNDM blaOXA, blaIMP, five and two different isolates harbored blaNDM and blaOXA, and blaOXA and blaIMP respectively. The Meropenem, Imipenem and Ertapenem MIC values for the isolates ranged from 2g/mL to ≥256g/mL. Phylogenetic grouping showed that the CRE E.coli isolates belonged to five different groups; groups A, B1, C, D and unknown. The detection of carbapenem resistant E.coli in this study shows that CRE is has become an emerging problem in farm animals, particularly, in poultry farms. This also implies the potential public health risks posed by CRE from chicken to the consumers.


Introduction
Carbapenem resistance in Enterobacteriaceae is a serious emerging antimicrobial resistance (AMR) issue that has been escalating and posing challenges in treating infections caused by the with fever, septicaemia, pneumonia, peritonitis, meningitis, and device-associated infections [1]. The bacteria in this family are transmitted easily between human and animals, especially via fomites, food and water. During the transmission, genetic materials are transferred through horizontal gene transfer, mediated mostly by plasmids and transposons. Enterobacteriaceae are among the common nosocomial pathogens often causing infections through medical devices that include ventilators, intravenous catheters, urinary catheters, or wounds caused by injury or surgery [2]. Such nosocomial infections commonly affect immunocompromised patients and in patients being treated using invasive devices. Carbapenem is a broad spectrum Beta lactam antibiotic that is regarded as the last-line antibiotic, especially to be used in critically ill patients who have developed antimicrobial-resistant bacterial infection. Unfortunately Enterobacteriaceae have developed resistance against this last resort drug and made it ever challenging to treat infections caused by diseases caused by these carbapenem resistant Enterocateriaceae (CRE). Among the bacteria in the family Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae are the most commonly detected CRE that has been posing threat to the public health and animal health [3]. Such prevailing AMR issue has been compromising the efficacy of antibiotics and according to the World Health Organization, there is a possibility for the world to encounter an era, in which all the antibiotics become ineffective thereby increasing mortality rate and increasing cost of treatment if no intervention is done to overcome the problem. There are also concerns that failure to counter the rising AMR problems worldwide may lead to re-emergence of previously eradicated or controlled diseases [4].
According to the National Surveillance of Antimicrobial Resistance (NSAR) in Malaysia from 2006 to 2017, which analysed the data obtained from hospital microbiology laboratories from different parts of the country, carbapenem resistance in E. coli declined from 0.5% in 2010 to 0.2% in 2014 [5].
However, there are no recent and comprehensive studies conducted on the prevalence of CRE in the country, particularly in agricultural sectors including farm animals and animal products. Moreover the data on the phylogenenetic diversity, antimicrobial resistance profiles and diversity in carbapenemase resistance encoding genes in E. coli from food animals, particularly chicken are scarce. Therefore this study was conducted to detect the presence of carbapenem resistant E.coli in live chicken, investigate the antimicrobial resistance patterns, determine the phylogeny and identify the common carbapemase genes in carbapenem resistant E.coli isolates from live chicken.

Bacterial isolation, identification and antimicrobial resistance patterns
Based on the routine microbiology, 56.67% (

Phylogenetic analyses
The results from quadriplex PCR showed that the CRE E.coli belong to diverse phylogroups including, group A, groupB1, group C, group E, group D and group unknown. Among the 19 CRE isolates, nine were identified as members of group A while five, three and one were respectively typed as group B1, group C, group D and unknown group (figure 2 and table 1).

Discussion
Antibiotic resistance is a global public health concern and the continuous emergence and spread of resistant bacteria has compounded the challenges in treating infections caused by antibiotic resistant bacteria. Carbapenem resistance in common bacterial pathogens has become one of the most concerning global public health issues since the carbapenem antibiotics are among the most critically important antimicrobials for treatment of infections in humans [6]. Carbapenems have been reported to show the broadest spectrum of antimicrobial activity in vitro against Gram-positive and Gram-negative bacteria, including anaerobes [7]. Because of their broad spectrum of actions, potency and effectiveness in treating broad range of infections in human, carbapenems have been recognized as the antibiotics of last resort to treat infections caused by multidrug-resistant Gram-negative bacteria [8]. Although Carbapenemases have been known to be new and potentially emerging problem in food-producing animals, the prevalence of carbapenem resistance in bacteria from animals have been scarcely reported [9]. So far most of the prevalence rate of 15% and 6% respectively from the broilers water samples. Among the poultry CRE isolates (n=15), all of were blaNDM positive, while blaKPC, blaOXA48 and blaNDM genes were detected in 11 of the isolates while four isolates were positive for either blaKPC or blaNDM or blaOXA-48 and blaNDM. The same study also reported a high prevalence, 56% of K. pneumoniae isolates from humans harbouring multiple genes [10]. This finding suggests that a high incidence of CP K. pneumonia in humans may contribute to its dissemination among food-producing animals and the livestock environment, thus increasing the risk of foodborne transmission to the consumers [10]. The presence of carbapenem resistance in bacteria from animals, including food-producing animals (pigs, bovines and horses) has also been reported from some European countries such as Germany, France and Belgium [9]. The identification of E.coli isolates harbouring multiple (at least two) carbapenemase encoding genes from food animal in this study differentiates it from previous similar studies which mostly reported E. coli isolates harbouring one or two carbapenemase genes [11,12].
Carbapenems are not routinely used in food animal production including poultry farming; however, carbapenem resistance in the E.coli isolates might have coevolved along with resistance to other antibiotics that are commonly used in resistant strains of bacteria may also be disseminated through direct contact, insect vectors, and other animals [8,13,14]. An earlier study by Poirel et al. [15] also suggested that co-selection of carbapenemase genes under the selection pressure imposed by the use of aminopenicillins and aminopenicillin-β-lactamase inhibitor combinations in livestock may lead to the emergence and spread of carbapenem resistance. Reports from previous studies indicated that CRE can persist in animal production if the bacteria are adapted to animals and the farm environment and are stabilized by co-expression of further resistance genes [16,17]. The possibility that infected or carrier humans, particularly the farm workers might spread resistant bacteria in farms through direct and direct routes of transmission cannot be ruled out. This is due to the fact that humans, the farm workers in the context of the current study are more likely to have been exposed to broad-spectrum antibiotics, and in particular to broad-spectrum b-lactams, than the chickens [14]. Since CRE can transmit through ddirect anthropozoonotic or zooanthroponotic routes [18], the spread of CRE in humans may pose risk for food animal production and possibly lead to the establishment of CRE in the food animal production ecosystem and may lead to subsequent further spread of these pathogens [16].

Ethics
This After incubation, zone of inhibition for each of the antibiotic discs was measured and the antibiotic susceptibility was determined based on CLSI guidelines [25].

Determination of Minimum Inhibitory Concentration (MIC) using E-Test
The MIC determination using E-test (Biomerieux, France) was done as recommended by the manufacturer. Briefly, overnight culture of E.coli was suspended in 10 mL normal saline (0.9% NaCl). The turbidity of the bacterial suspension was adjusted to that of 0.5% McFarland standard.
The bacterial suspension was then uniformly streaked onto the entire surface of MHA (Oxoid, UK).
E-test strips (Biomerieux, France) interpretations of the results were done according to the CLSI standards [25]. Escherichia coli ATCC 25922 strain was used as quality control.

DNA Extraction
Following bacterial isolation and identification, genomic DNA extraction was performed for all the presumptive E.coli isolates using boiling method. One to two bacterial colonies from each of the isolates on Nutrient agar were re-suspended into a 1.5 ml microcentrifuge tube containing 100 µL of 10 mmol/L Tris-HCl buffer (pH 8.0). The microcentrifuge tubes containing the samples were vortexed and spun. The suspensions were then boiled for 10 minutes to lyse the cells, followed by quickly chilling on ice for 5 minutes. Then, the tubes containing the suspensions were centrifuged at 12000rpm for 10 minutes. Following that, 100 µL of the supernatant containing DNA from each of the microcentrifuge tubes were transferred into another 1.5 ml microcentrifuge and the DNA quality was assessed using spectrophotometer and gel electrophoresis and DNA extracts with acceptable qualities were stored at -20˚C until further use.

Molecular detection of E.coli and carbapenem resistance encoding genes
PCR amplification was conducted to identify E.coli using primers Pho-F/Pho-R targeting the housekeeping genes of E.coli and carbapenemase encoding genes (blaNDM, blaOXA, blaIMP, blaKPC) as described earlier [1,26].

Phylogenetic Analysis
Characterization of the phylogenetic groups of the E. coli isolates was determined according to the protocols described by Clermont et al. [8]. Briefly, a single PCR reaction mixture containing 12. supplied by Integrated DNA Technologies (Singapore). PCR amplifications were carried out in a Nexus gradient Mastercycler (Eppendorf, USA) using the following conditions: initial denaturation at 94 ∘ C for 4min and 30 cycles for each denaturation at 94 ∘ C for 5sec annealing at 57 ∘ C for 20sec (group E) or 59 ∘ C for 20sec (quadruplex and group C), amplification at 72∘C for 1min, and final extension at 72 ∘ C for 5min. The PCR products were analyzed by electrophoresis in 1.5% agarose gel and image analysis was done using GelDoc TM EZ Imager (Bio-Rad, USA). trpBA.r 5′-GCAACGCGGCCTGGCGGAAG-3′

Conclusions
In conclusion, the detection of carbapenem resistant E.coli in this study shows that these resistant bacteria are not limited to the hospital environment and that CRE is also an emerging problem in farm animals, particularly, in poultry farms. This may raise concern that these carrier