key: cord-0851821-oa87gc7r authors: Bendjama, Esma; Loucif, Lotfi; Chelaghma, Widad; Attal, Chahrazed; Bellakh, Fatma Zohra; Benaldjia, Randa; Kahlat, Imène; Meddour, Amna; Rolain, Jean-Marc title: First detection of OXA-48-producing Enterobacter cloacae isolate from currency coins in Algeria date: 2020-09-20 journal: J Glob Antimicrob Resist DOI: 10.1016/j.jgar.2020.09.003 sha: 5123fef0fba577dc3165e28edda263dfffd9592d doc_id: 851821 cord_uid: oa87gc7r OBJECTIVES: The aim of this study was to screen for the presence of β-lactamases-producing Gram negative bacteria isolates from Algerian currency collected from food vendors in Batna city, Algeria. METHODS: During two periods, May 2018 and from March to April 2019, a total of 408 coins and notes currency of different denominations of Algerian Dinar were randomly recovered from some food vendors. Samples were subjected to selective isolation of extended spectrum cephalosporins and carbapenem resistant Gram negative bacteria. The bacterial species identification was performed using Matrix-Assisted Laser Desorption–Ionization Time-of-Flight Mass Spectrometry. Antibiotic susceptibility testing was performed using the disk-diffusion method. Carbapenemases and extended-spectrum β-lactamases genes were searched for by Real-time PCR, Standard PCR and sequencing. The clonality relationships of carbapenemase producing isolates were investigated by multilocus sequence typing. The transferability of the detected carbapenemase encoding gene was verified by conjugation experiments. RESULTS: A total of twelve cefotaxime and/or carbapenem-resistant strains were isolated in this study and identified as E. cloacae, Raoultella ornithinolytica, Citrobacter freundii, E. coli, P. aeruginosa, Pseudomonas libanensis and Pseudomonas stutzeri. The bla(OXA-48) gene was detected in only one E. cloacae strain belonging to the ST108, while the two Raoultella ornithinolytica isolates harboured the bla(CTX-M-27) gene and one E. coli strain carried the bla(CTX-M-14) gene. The detected bla(OXA-48) gene was transferable by conjugation. CONCLUSION: In this study, we report for the first time, the detection of OXA-48-producing Enterobacter cloacae isolate from money. Thus, it calls for consciousness development on the potential risks associated with poor handling of currency. Currency is used as a medium for goods, exchange and services in the entire global economic environment and has promoted trade worldwide [1;2] . The potential threat to public health that money might act as a vehicle for the transmission of potential pathogenic microorganisms was suggested in the 1970s by Abrams and Waterman [3] , given that money is handled and circulated by a large number of people, under different environmental and personal conditions [1;2] . Consequently, recent studies from different parts of the world have confirmed these theories and have shown that viable pathogenic organisms can be isolated on the money surfaces, including bacteria, particularly Gram negative which may persist more than Gram positive on inanimate surfaces and viruses such as human influenza virus and Coronavirus [2;4] . In addition, various studies show that currency could play a major role in the spread of bacteria resistant to commonly used antibiotics and thus pose a threat to the public health of currency handlers and to the community [2] . The β-lactam antibiotics have been amongst the most effective and widely used drugs for the treatment of bacterial infections in both human and veterinary medicines. In the past decade, J o u r n a l P r e -p r o o f resistance to β-lactams group in Gram negative bacteria (GNB) was mainly due to extendedspectrum β-lactamases (ESBLs) and plasmid-encoded AmpC-type cephalosporinases production. Furthermore, these determinants have been recognized to be of growing importance worldwide. Thus, carbapenems have become among the last-resort drugs in the treatment of severe infections caused by these bacteria. Unfortunately, widespread use of carbapenems has led to the emergence of carbapenem-resistant GNB [5;6] . Carbapenem resistance involves multiple mechanisms. However, the most common one is the production of carbapenemases. Carbapenem-hydrolyzing beta-lactamases are diverse enzymes, belonging to the different Ambler classes A, B or D, among which OXA-48 class D carbapenemase is mostly identified in enterobacterial species. OXA-48 hydrolyses carbapenems only at a low level, but not broad-spectrum cephalosporins [6] . The blaOXA-48 gene was first identified in a clinical K. pneumoniae strain isolated in Istanbul, Turkey in 2001. Since then, the OXA-48 enzyme has become endemic in many areas, particularly in the Mediterranean region, especially North African countries [7] , where Algeria is known by the huge spread of this gene from different sources, including the community setting. In most parts of the world, it is believed that the simultaneous handling of food and money contributes to the frequency of food borne infectious agents, thus risking the safety of consumers in the community [1;8;9] . To the best of our knowledge, no studies have been published on carbapenemase-producing Gram negative bacteria from currency and no data were available. However, the World Health Organization recommends that more studies be conducted in different sites, particularly in African countries to assess the public health effects of money contaminated by micro-organisms [3] . Therefore, the aim of this study was to screen for the presence of β-lactamases-producing Gram negative bacteria isolated from Algerian currency collected from food vendors in Batna city, Algeria. The two sides of each currency note were wiped using a sterile cotton swab pre-dipped in brain-heart infusion (BHI) broth (BD: Becton, Dickinson and company, France) supplemented with Tween 80 solution at 0.05% to ensure that the microbial content was dislodged. Swabs used for the same note category collected from the same market and at the same period were pooled (Two to four notes per pool) and pre-enriched for 4 hours at 37°C in BHI broth. Currency coins were directly pre-enriched in bottles under the same conditions (eight to ten coins per pool). Thereafter, 1 ml of each pre-enriched sample was transferred in three tubes of 10 ml BHI broth supplemented with 64μg/ml of vancomycin and three different antibiotics: (i) 2μg/ml of cefotaxime, (ii) ertapenem (2μg/ml) and (iii) imipenem (9 μg/ml), respectively. After overnight incubation at 37°C, a volume of 10 μl aliquot from positive tubes were The obtained isolates were phenotypically tested for ESBL production by the double-disk synergy test (DDST). The modified Carba NP test (MCNP test) and the modified Hodge test were applied to screen for the carbapenemase activity [6] . The strains were analyzed by realtime PCR, further verified by standard PCR and sequencing using specific primers for ESBLencoding genes (blaTEM, blaSHV and blaCTX-M) and for carbapenemase genes (blaNDM, blaVIM, blaKPC and blaOXA-48). Transferability of the blaOXA-48 gene was performed using conjugation experiments between the donor isolate and sodium azide resistant Escherichia coli J53 as recipient strain. We separately inoculated the recipient isolate and donor carrying blaoxa-48 gene overnight into BHI broth (10 mL), and then mixed the samples at a ratio of 1:10 (donor: recipient) incubated for 4 hours. After that, 10 μl of the resulting mixture were inoculated on nutrient agar plates containing 200 μg/ml sodium azide and 2 μg/ml ertapenem and incubated for 18 to 24 hours J o u r n a l P r e -p r o o f at 37°C. The obtained transconjugant was subjected to antimicrobial susceptibility testing, the MCNP test and the modified Hodge test. Positive carbapenemase production was confirmed by OXA-48-PCR and sequencing. Multilocus sequence typing (MLST) was performed for the OXA-48-producing E. cloacae isolate by targeting seven housekeeping genes (dnaA, fusA, gyrB, leuS, pyrG, rplB and rpoB), the MLST data were analyzed through the PubMLST scheme (https://pubmlst.org/ecloacae/). A total of twelve cefotaxime and/or carbapenem-resistant isolates were obtained from six currency coins pooled samples as presented in table 1. However, none of them were isolated from currency notes. Out of the 12 isolates, nine were Enterobacteriaceae (9/12) and three were nonfermenting bacteria (3/12). The obtained isolates were identified as follows: E. cloacae (4 strains), Raoultella ornithinolytica (2 strains), Citrobacter freundii (2 strains), E. coli (1 strain), P. aeruginosa (1 strain), Pseudomonas libanensis (1 strain) and Pseudomonas stutzeri (1 strain). Antibiotic susceptibility tests showed that all twelve strains are resistant to almost all of the antibiotics tested as follows: amoxicillin (12/12), cefoxitin (9/12), cefotaxime (12/12), ceftazidime (7/12), cefepime (1/12), aztreonam (2/12), amoxicillin-clavulanic acid (8/12), J o u r n a l P r e -p r o o f ticarcillin-clavulanic acid (8/12), ertapenem (4/12), imipenem (3/12), tobramycin (2/12) and ciprofloxacin (4/12), but remained susceptible to gentamicin and amikacin. Three isolates were positive for the double-disk synergy test (2 Raoultella ornithinolytica and one E. coli). The modified Hodge test and the modified carba NP test were positive only for one E. cloacae strain, while no carbapenemase activity was detected in three carbapenem-resistant Pseudomonas isolates. Antibiotic susceptibility testing results and those of the phenotypic characterization of β-lactamase production are summarized in Table 1 . Screening for ESBL and carbapenemase-encoding genes demonstrated that two R. Table 1) . Multilocus sequence typing analysis showed that the OXA-48-producing E. cloacae isolate belonged to the sequence type 108. Conjugative transfer of blaOXA-48 gene into the E. coli J53 strain was successful, as the transconjugant obtained on the selective medium containing sodium azide and ertapenem was MCNP test positive. The transferability of the carbapenemase blaOXA-48 gene was confirmed by PCR and sequencing. To the best of our knowledge, this is the first report of OXA-48 carrying Enterobacteriaceae from currency. The role of money in the transmission of infection is not a new concept. It dates back from 1665 in England, when coin currency was commonly assumed to be one of the vehicles for the diffusion of plague that killed more than 60,000 people [10] . Studies concerning the contamination of coins and paper have been few and rather limited. However, after the outbreaks of some transmissible diseases, these reports have gained some serious consideration [11;12] and revealed that coins and paper currencies can serve as an ideal breeding for the multiplication of microorganisms [1] . In this study, we isolated different Gram negative bacteria species in agreement with several studies carried out on coin and paper currency collected from different sites including hospitals, public transport conductors and food vendors among different countries [1-3;9;13] . Most of the bacterial species identified here have been implicated in many life threatening infections, which could pose a health risk particularly to immune compromised individuals in the community. In addition, to P. libanensis detected here for the first time from currency, the other GNB species have been already described with different levels of antibiotic resistance [3;8;9;13;14] . The obtained isolates could be from several sources depending on environmental conditions, age of money, handling or the poor sanitary condition and hygienic practices exercised by those manipulating currencies [2;3] . The detection of coliforms such Escherichia coli, which can survive 11 days on the inert surfaces of utilities, could suggest the fecal contamination of coins currency as a result of the poor personal hygiene [1] . Banknotes offer a large surface to be contaminated by microorganisms, which can persist on it for longer periods. In addition, the surface of paper currency is irregular, thus facilitating adherence by different types of microorganisms [3] . However, in our study, none of the antibiotic-resistant strains were isolated from our Algerian paper based banknotes, which could be explained by the limited number of the analyzed note samples (31 samples) and the absence or the low bacterial load J o u r n a l P r e -p r o o f of antibiotic-resistant Gram negative strains as well as the age of the paper collected, since newer paper notes contain less microbes [2;3] . The Algerian coins are composed of nickel, AISI 430 steel and cooper, these compounds have been shown to possess antimicrobial activity, which may explained the low rate of bacterial contamination obtained in our study [3;15] . There are several investigations confirming that antibiotic-resistant bacteria may contaminate money and that this latter might play an important role in their widespread. Few studies had focused on antibiotic resistance among Gram negative bacteria recovered from currency. In our study, we followed the selective isolation approach, which allowed us to isolate 12 drug- pneumoniae, E. cloacae, Pseudomonas aeruginosa and Acinetobacter baumannii from banknote has been published in Sudan, where at the difference of our study, they evaluated the bacterial contamination rate of 135 banknotes showing a contamination rate of 100% [14] . The blaCTX-M-27 gene was first described in E. coli strain isolated in 2001 from a patient hospitalized in Grenoble, France, this strain co-harbored the blaTEM-1 gene [16] . After this report, several studies showed that CTX-M-27 is emerging worldwide, especially in Europe and Japan [17] . Furthermore, the blaCTX-M-14 gene has already been detected in multi-drug resistant E. coli isolated from fresh ground beef in Algiers [18] . Recently, Nabti et al. reported the isolation of four E. coli strains that co-harbored the blaCTX-M-14 and blaOXA-1 ESBL genes or the blaCTX-M-14 with blaTEM-1 β-lactamases genes from outpatients and inpatients in Setif University Hospital [19] . Worryingly, our study is the first to report the detection of pOXA-48-producing E. cloacae from currency coin. Moreover, it is of special interest that OXA-48 like enzyme has been identified in Algeria, a part of North Africa, where OXA-48-type carbapenemase is considered endemic. Indeed, various studies on OXA-48-like-producing Enterobacteriaceae have been published in Algeria, involving clinical strains recovered from various hospitals located in different regions in Algeria including outbreaks [6;7] , from companion and wild animals, river water and from fresh vegetables [7] . More recently, the blaOXA-48 gene has also been detected in Algerian community [5] . The OXA-48-producing E. cloacae isolate identified in the current study was shown to belong to the sequence type 108. This high-risk international clone (ST108) has been previously associated with CTX-M-9 and SHV-12 co-production, TEM-3 and SHV-12 production in clinical E. cloacae strains recovered from different European countries including France, Greece, Italy and Luxembourg [20] . In addition, this study reports the first detection of E. cloacae ST810 clone in Algeria. In conclusion, our findings showed that Algerian currencies may play an important role in the transmission of carbapenem resistant Gram negative bacteria, particularly those producing the silent menace OXA-48. These data constitute a starting point; further researches are nevertheless needed to determine the potential role of currency in the dissemination of these high levels of resistance to β-lactams in the community. Thus, it calls for consciousness development at all levels, especially among personnel working in food establishments and on the possible health risks associated with poor handling of currency. Microbial load and safety of paper currencies from some food vendors in Jimma Town, Southwest Ethiopia Microbial contamination of currency notes and coins in circulation: A potential public health hazard Public health implications of contamination of Franc CFA (XAF) circulating in Buea (Cameroon) with drug resistant pathogens How long do nosocomial pathogens persist on inanimate surfaces? 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