Clonal Dissemination of Plasmid-Mediated Carbapenem and Colistin Resistance in Refugees Living in Overcrowded Camps in North Lebanon

Carbapenem and colistin-resistant bacteria represent a global public health problem. Refugees carrying these bacteria and living in inadequate shelters can spread these microorganisms. The aim of this study was to investigate the intestinal carriage of these bacteria in Syrian refugees in Lebanon. Between June and July 2019, 250 rectal swabs were collected from two refugee camps in North Lebanon. Swabs were cultured on different selective media. Antibiotic susceptibility testing was performed using the disk diffusion method. Carbapenemase-encoding genes and mcr genes were investigated using real-time polymerase chain reaction (RT-PCR) and standard polymerase chain reaction (PCR). Epidemiological relatedness was studied using multilocus sequence typing (MLST). From 250 rectal swabs, 16 carbapenem-resistant, 5 colistin-resistant, and 4 colistin and carbapenem-resistant Enterobacteriaceae were isolated. The isolates exhibited multidrug-resistant phenotypes. Seven Klebsiella pneumoniae isolates harboured the blaOXA-48 gene, and in addition four K. pneumoniae had mutations in the two component systems pmrA/pmrB, phoP/phoQ and co-harboured the blaNDM-1 gene. Moreover, the blaNDM-1 gene was detected in six Escherichia coli and three Enterobacter cloacae isolates. The remaining five E. coli isolates harboured the mcr-1 gene. MLST results showed several sequence types, with a remarkable clonal dissemination. An urgent strategy needs to be adopted in order to avoid the spread of such resistance in highly crowded underserved communities.


Introduction
Antimicrobial resistance (AMR) has increased markedly in recent years. This is particularly important when facing complex infections in humans [1]. AMR is expected to result in severe mortality and economic losses that will increase the cycle of poverty [2]. This global public health concern causes huge clinical and economical losses, mainly in developing countries [3]. Thus, the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) have recognised AMR as an urgent and global crisis and threat [4,5].
Carbapenems are a group of β-lactam drugs that are often used as last resort antibiotics to treat infections arising from multidrug-resistant (MDR) Gram-negative bacteria [6]. Unfortunately, carbapenemase-producing Gram negative bacteria have become a major concern around the world [7][8][9]. They are a growing concern because they confer resistance Between June and July 2019, 250 rectal swabs were collected from the inhabitants of two Syrian refugee camps, located in North Lebanon, in the Akkar Governorate ( Figure 1). One hundred random samples were collected from the Talhayat camp and another 150 from the Bebnine camp. The swabs were grouped according to whether they came from residents of the same tent and/or family. The swabs were inserted through the rectal sphincter 1-1.5 inches (2-3 cm) and rotated gently. Tubes containing the swabs were placed in a refrigerator (2-8 °C), after checking that faecal material was visible on the tip of the swab. The swabs were directly shipped to the laboratory (less than 1.5 h away) where they were directly cultivated on Tryptic soy broth (TSB) medium (Becton Dickinson GmbH, Heidelberg, Germany).

Bacterial Identification
The rectal swabs were cultivated in the growth medium Tryptic soy broth (TSB) (Becton Dickinson GmbH, Heidelberg, Germany) and incubated for 24 h at 37 °C. Different selective media, including Lucie Bardet Jean-Marc Rolain (LBJMR) [29] and MacConkey agar (bioMérieux, Marcy l'Etoile, France) supplemented with Ertapenem (2 μg/mL) were then used to culture 100 μL of the enrichment medium. These media were used to screen colistin resistant and carbapenem resistant organisms, respectively. The selected isolates were then sent to the laboratory in Marseille, France. Bacterial identification at the species level was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) (Microflex; Bruker Daltonics).

Bacterial Identification
The rectal swabs were cultivated in the growth medium Tryptic soy broth (TSB) (Becton Dickinson GmbH, Heidelberg, Germany) and incubated for 24 h at 37 • C. Different selective media, including Lucie Bardet Jean-Marc Rolain (LBJMR) [29] and MacConkey agar (bioMérieux, Marcy l'Etoile, France) supplemented with Ertapenem (2 µg/mL) were then used to culture 100 µL of the enrichment medium. These media were used to screen colistin resistant and carbapenem resistant organisms, respectively. The selected isolates were then sent to the laboratory in Marseille, France. Bacterial identification at the species level was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) (Microflex; Bruker Daltonics).

Phenotypic Detection of Carbapenemase Activity
Three phenotypic tests, the modified Hodge test (MHT), the modified Carba NP test (MCNP), and the ethylene diamine tetra-acetic acid (EDTA) test, were performed to detect the production of carbapenemase, as described previously [30,31].

DNA Extraction
The extraction of the bacterial DNA was performed by the EZ1-automatic robot (Qiagen Biorobot EZ1-, Tokyo, Japan) using an extraction kit (DNA EZ1, Qiagen, Hilden, Germany), following the manufacturer's instructions. The extracted DNA was kept at −20 • C. In order to determine the genetic location of the carbapenemase and mcr genes, plasmid extraction was performed for the isolates that showed a positive PCR result for these genes using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, Waltham, MA, USA).
Standard PCR and sequencing of antibiotic resistance genes was performed on an automated ABI 3130 sequencer (PE Applied Biosystems, Foster City, CA, USA) using BigDye terminator chemistry. The sequenced genes were analysed using BlastN and BlastP and then compared with the ARG-ANNOT database.
In order to determine whether the recognised amino acid substitution resulting from a missense mutation could affect the function of the protein, PROVEAN (Protein Variation Effect Analyser) software (http://provean.jcvi.org/seq_submit.php, accessed on 18 February 2021) was used. If the score of the variant proteins was below or equal to a predefined threshold (−2.5), it was considered to have a "deleterious" effect, and if the score was above the threshold, it was predicted to have a "neutral" effect.

Multilocus Sequence Typing (MLST)
In order to determine the clonal relationship of the isolates, the MLST method was performed as described on the Institute Pasteur's MLST website (www.pasteur.fr/mlst, accessed on 23 March 2021).
Due to the absence of mcr genes in K. pneumoniae ColiR (Table 2), genes implicated in colistin resistance (mgrB, pmrA, pmrB, phoP, and phoQ) were amplified and sequenced. As shown in Table 3, the result of the sequence analysis showed that there was no mutation in the mgrb, pmrA, and phoP genes for the four colistin-resistant K. pneumoniae isolates. These isolates were resistant to colistin due to mutations in the pmrB and phoQ genes (Table 3). For the colistin resistant Kp-1 isolate, the analysis revealed a nucleotide deletion (C577del) in the pmrB gene, leading to a frameshift mutation and resulting in a defective protein.
For the colistin resistant Kp-2, the analysis revealed a nucleotide insertion (G432_C433insG) in the pmrB gene, causing a frameshift mutation and resulting in a defective protein. For the Kp-3 isolate, the analysis also revealed a nucleotide insertion at position (T459_G460insC) in the phoQ gene leading to a frameshift mutation and resulting in a defective protein.

Discussion and Conclusions
MDR bacteria are now considered as a serious public health problem due to the limited range of antibiotics used to treat infections caused by these bacteria. The antimicrobial resistance crisis is mainly due to the misuse of these antibiotics in several sectors (human and animal) and the luck of discovery of new drugs [38]. Refugees coming from countries with a high occurrence of antimicrobial resistance can introduce the resistant isolates to their new country of residence. These refugees can spread these resistant bacteria, especially if they are living in shelters with poor living conditions. This study describes the intestinal carriage of carbapenem-and colistin-resistant bacteria particularly in Enterobacteriaceae among healthy Syrian refugees living in two shelters (Bebnine and Talhayat) in North Lebanon.
All carbapenem-resistant E. coli isolates (six isolates) harboured the bla NDM-1 gene. Five of them (EC-1, EC-2, EC-3, EC-4, and EC-5) were collected from the same shelter (Bebnine), and from the same tent; three isolates (EC-1, EC-2, and EC-3) belonged to ST361 and two (EC-4 and EC-5) belonged to ST1294. The bla NDM-1 E. coli ST361 had already been reported in hospitalised patients in South Korea [39]; however, ST1294 is mainly found in animals and in the environment [40][41][42][43][44][45]. These results shed light on the clonal dissemination of these isolates between refugees, since the same clones were found in more than one person in the same tent, but also to the probable zoonotic or environmental origin of the ST1294 isolates. The sixth isolate (EC-6) belonged to ST648 and was collected from the Talhayat shelter. This clone was previously reported in different studies in hospitalised patients [46][47][48]. In Lebanon, NDM-1 producing E. coli isolates were first described in 2012 in Iraqi patients referred to Lebanon [49]. Moreover, NDM-1 producing E. coli was also reported in 2013 at a tertiary care centre in Lebanon [50]. To the best of our knowledge, this is the first report of intestinal carriage of NDM-1 producing E. coli in healthy individuals.
All carbapenem-resistant K. pneumoniae isolates (seven isolates: KP-2, KP-3, KP-4, KP-5, KP-6, KP-7, and KP-8) harboured the bla OXA-48 gene, of which three also had the bla NDM-1 gene (KP-6, KP-7, and KP-8). The carbapenem-resistant K. pneumoniae isolates (KP-2, KP-3, KP-4, and KP-5) harbouring the OXA-48 gene had the same ST (ST16) and were collected from the same shelter (Bebnine) and from closely related tents. OXA-48 producing K. pneumoniae ST16 was previously described in clinical isolates in Spain [51]. In Lebanon, several studies have reported the occurrence of OXA-48 producing bacteria and OXA-48 producing K. pneumoniae, all from hospital settings [49,[52][53][54]. In addition, the remaining three isolates (KP-6, KP-7, and KP-8) harbouring both the NDM-1 and the OXA-48 genes, also had the same ST (ST14), and were collected from the Talhayat shelter from closely related tents. NDM-1 and/or OXA-48 producing K. pneumoniae ST14 have been reported worldwide [55][56][57][58], highlighting the rapid spread of this clone. In Lebanon, several studies have described NDM-1 and/or OXA-48 producing K. pneumoniae isolates [49,52,53]; however, this is the first description of the co-occurrence of NDM-1 and OXA-48 genes in the same isolate, which highlights the dissemination of plasmids carrying these resistance genes between isolates. Furthermore, results showed that K. pneumoniae isolates recovered from closely related tents in the same shelter belonged to the same clone. Syrian refugees in Lebanon live in inadequate shelters living in crowded conditions with poor sanitary facilities, which increases the spread of resistant bacteria. This explains the dissemination of the same clone in refugees living in closely related tents within the same shelter.
All carbapenem-resistant E. cloacae isolates (Eclo-1, Eclo-2, Eclo-3) harboured the bla NDM-1 gene, of which two (Eclo-1 and Eclo-2) were isolated from the same tent at the Bebnine shelter and belonged to the same clone (ST182). The remaining isolate belonged to ST1120 and was isolated from the Talhayat shelter. NDM-1 producing E. cloacae ST182 have been reported in many countries, including China, Czech Republic, and Kenya [59,60]. This clone is of high concern because of its ability to disseminate rapidly, causing outbreaks as was the case in Mexico [61]. Hence, controls should be performed to avoid the rapid dissemination of such clones. To the best of our knowledge, this is the first detection of NDM-1 producing E. cloacae in Lebanon.
All colistin-resistant E. coli isolates harboured the mcr-1 gene, of which three (EC-7, EC-8, EC-9) belonged to the same clone ST2001 and were isolated from closely related tents at the Bebnine shelter. The remaining E. coli isolates belonged to ST101 (Bebnine shelter) and ST4187 (Talhayat shelter). E. coli ST2001 has been previously described in feacal samples from wild animals [62] and in an organic broiler farm [63]. ST4187 has been described in broiler chickens in Tunisia [64], thus pointing towards a zoonotic origin of our isolates, since the refugees are in close contact with wild animals. The ST101 clone has been reported in clinical samples from Brazil and Iran [65,66]. In Lebanon, E. coli harbouring the mcr-1 gene has been described in few studies in hospitalised patients [67], however, no other study has highlighted its presence in healthy individuals. To the best of our knowledge, this is the first description in Lebanon of the intestinal carriage of E. coli isolates harbouring the mcr-1 gene in healthy individuals. This is alarming, since the plasmid carrying the mcr-1 gene could disseminate between species, making them resistant to one of our last resort antibiotics.
All colistin-resistant K. pneumoniae isolates harboured the bla NDM-1 gene and had mutations in the PmrB and/or PhoQ genes (Table 3). They were all isolated from the same family (in the Bebnine camp) and belonged to the same clone ST944. An ST944 clone harbouring the mcr-1 gene has been previously described in chicken meat from western Algeria [68]. A recent study conducted in Egypt [69] described colistin and carbapenem resistance in K. pneumoniae strains isolated from chicken and humans. The study showed that all K. pneumoniae were MDR, with 45.9% harbouring a ß-lactamase gene with a carbapenemase gene, 18.9% harbouring a ß-lactamase gene with the mcr-1 gene, and 13.5% harbouring a β-lactamase gene with both carbapenemase and mcr-1 genes. In Lebanon, few studies have described colistin-resistance in K. pneumoniae isolates such as those in 2015 [70] and 2017 [71]. Our study showed new mutations (Table 3) in the pmrB and/or phoQ genes resulting in a defective protein with a stop codon. Interestingly, a study conducted in Lebanon in 2018 [72] described colistin-resistance in clinical K. pneumoniae isolates due to mutations in the mgrB, pmrB, and phoQ genes. These isolates also harboured the bla NDM-5 gene. Our study reported for the first time in Lebanon, colistin-resistance K. pneumoniae isolates with new mutations in pmrB and/or phoQ genes and harbouring the bla NDM-1 gene.
In conclusion, the intestinal carriage of MDR bacteria in community settings is of great concern. Our study reported for the first time the intestinal carriage of carbapenemand/or colistin-resistant isolates in refugees, which is alarming since plasmids carrying resistance genes can spread between bacteria and refugees within the same shelter. Syrian refugees come from a country with a high prevalence of antimicrobial resistance where antibiotics can be taken without prescription, thus they can spread MDR bacteria to their final destination, in this case, Lebanon. In addition, these refugees are living in inadequate shelters which can contribute to the spread of these bacteria. Molecular and epidemiological studies are essential in order to better understand the mode of transmission of these microorganisms. An urgent strategy must therefore be adopted to limit the spread of these MDR bacteria within the community.

Data Availability Statement:
The data presented in this study are available in Tables 1 and 2.