Emergence of carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter baumannii in livestock animals in Lebanon

Sir, Infections caused by carbapenemase-producing bacteria are a serious public health problem related to antibiotic resistance.1 A worrying new problem is the dissemination of these organisms in livestock animals. Here, we report the detection of VIM-2-producing and OXA-23-producing Pseudomonas aeruginosa and Acinetobacter baumannii, respectively, in livestock animals from Lebanon. We also detected the co-occurrence of blaOXA-23 and blaOXA-58 in the same isolate of A. baumannii. During a 4 month study period in 2013, faecal specimens (n = 115) were collected from livestock animals (pigs, fowl and cattle) from different farms in North Lebanon. All the samples were cultured on nutrient agar supplemented with ertapenem (1 mg/L). In total, nine isolates grew and were identified by MALDI-TOF MS, including five A. baumannii and four P. aeruginosa. The results of antibiotic susceptibility testing revealed that the isolates were resistant to almost all antibiotics (Table S1, available as Supplementary data at JAC Online) and showed high-level resistance to carbapenems, with MICs of imipenem >32 mg/L. The isolates were then analysed by the modified Hodge test, MALDI-TOF MS Ultraflex assay, biochemical (CarbaNP and CarbAcineto) test and EDTA disc test as previously described,2 and all isolates gave positive results on the modified Hodge test and Carba test. In addition, the MALDI-TOF MS Ultraflex assay was positive for all the isolates, suggesting carbapenemase production. However, the activity of carbapenemase was inhibited by EDTA only in P. aeruginosa isolates, indicating that these isolates, but not those of A. baumannii, produced metallo-β-lactamases (Table 1). Phenotypic and genotypic features of the P. aeruginosa and A. baumannii isolates PA, P. aeruginosa; AB, A. baumannii. Phenotypic and genotypic features of the P. aeruginosa and A. baumannii isolates PA, P. aeruginosa; AB, A. baumannii. Screening for carbapenemase-encoding genes by PCR showed that all the P. aeruginosa isolates harboured the blaVIM-2 gene and all the A. baumannii isolates harboured the blaOXA-23 gene. One of the A. baumannii isolates had the blaOXA-58 gene in addition to the blaOXA-23 gene (Table 1). The first description of carbapenemase-producing isolates from livestock was reported in Acinetobacter spp. producing OXA-23 in cattle in France.3 In addition, in China, a blaNDM-1-producing A. baumannii from a pig has been reported.4 The first carbapenemase-producing Salmonella isolated from livestock were reported in Germany, where the researchers detected the presence of blaVIM-1-producing Salmonella enterica subsp. enterica in pigs.5 This carbapenemase has previously been found in Germany in Escherichia coli on a pig farm.6blaVIM-2-producing P. aeruginosa has never been described to our knowledge in either livestock or companion animals despite the detection of blaVIM- and blaIMP-negative imipenem-resistant P. aeruginosa isolates from companion animals.7 In addition, this is the first co-occurrence of blaOXA-23 and blaOXA-58 genes in the same A. baumannii isolate in animals. All the conjugal transfer attempts, except for the isolate harbouring the blaOXA-58 gene, failed to yield E. coli transconjugants, suggesting that the OXA-23 and VIM-2 carbapenemases were chromosomally encoded and that the OXA-58 carbapenemase was plasmid mediated. In fact, the precise origins of these genes remain undefined, but they have all escaped from environmental bacteria into species with clinical relevance. For example, the blaOXA-48 family of enzymes occurs naturally in Shewanella spp., a genus that is found in lake sediments, and the OXA-23 carbapenemase originates from the environmental species Acinetobacter radioresistens.8 This indicates that these carbapenemase genes may have been mobilized from environmental bacteria into animals due to the close contact between the environment and the animals. The MLST analysis [as recommended by PubMLST (www.pubmlst.org/paeruginosa) and the Pasteur Institute (www.pasteur.fr/mlst)] showed that all the P. aeruginosa isolates had a new ST (ST1759, ST1760, ST1761 and ST1762). Three of the five A. baumannii isolates were also assigned to new STs (ST491, ST492 and ST493), while two of them belonged to ST2 and ST20, respectively. The A. baumannii ST2 clone has been detected in humans and was responsible for several outbreaks, such as the recent outbreak in Sicily,9 and the A. baumannii blaOXA-23 ST20 clone has been reported in horses and pets in Switzerland.10 Regarding these findings, we can speculate that animals may represent a reservoir for these genes since new clones that have never been seen in humans were detected in animals. For the ST2 clone, we believe that its presence in animals may not have been due to contamination from humans but due to a transfer from the environment to animals, the clone then being transmitted to humans due to possible links between animals and humans. Finally, while we cannot eliminate the possibility of human contamination, we cannot deny the evidence of an animal reservoir of these genes, especially as new clones were found in animals that have never been seen in humans. In conclusion, we report the emergence of P. aeruginosa producing VIM-2 carbapenemase and the co-occurrence of the blaOXA-23 and blaOXA-58 genes in the same A. baumannii isolate in livestock animals. These findings are of great concern because carbapenemases in bacteria from non-human sources are not yet being systematically investigated and, due to possible links with animals, humans may have high-risk factors for infection with such bacteria. This work was supported by the AZM Research Center for Biotechnology and its Application, Lebanese University, Lebanon. The research project was supported by the National Council for Scientific Research, Lebanon, and by IHU Méditerranée Infection and the French CNRS. None to declare. We would like to thank Linda Hadjadj, Taha Abdo and Maryam Yehya for their technical assistance.