Carbapenemase-producing Acinetobacter spp. in Cattle, France

To the Editor: Multidrug resistance in bacteria isolated from animals is an emerging phenomenon, mirroring what is happening among humans. During the past decade, expanded-spectrum β-lactamases in Enterobacteriaceae from humans (1) and animals (2) worldwide have been reported. Among humans, as a consequence of this high rate, use of carbepenems is increasing selection pressure; carbapenem-resistant gram-negative organisms are increasingly reported, including carbapenemase-producing Enterobacteriaceae and Acinetobacter spp (3). 
 
The most commonly acquired carbapenemases identified in Acinetobacter spp. correspond to carbapenem-hydrolyzing class D β-lactamases (3). In particular, the worldwide spread of OXA-23–producing A. baumannii is considered a serious threat; those strains are frequently involved in nosocomial outbreaks for which therapeutic options are extremely limited (3,4). Our study objective was to evaluate the possible occurrence of carbapenemase-producing gram-negative bacteria in dairy cattle in France. 
 
In August 2010, at a dairy farm 30 km from Paris, France, rectal swabs were collected from 50 cows. Samples were precultured in buffered peptone water and incubated for 18 h at 37°C. Cultures were inoculated by streaking 100 μL of the suspensions onto Drigalski agar plates (bioMerieux, Balmes-les-Grottes, France) containing 1 μg/mL of imipenem to select for carbapenem-resistant gram-negative isolates. Of the 50 samples, 9 produced growth on imipenem-containing plates. All colonies tested (10 colonies/sample) by using the API 20 NE (bioMerieux) system were first identified as A. lwoffii. Molecular techniques based on sequencing of the gyrA, gyrB, and rpoB genes (5) enabled more precise identification and indicated that all isolates belonged to the Acinetobacter genomospecies (DNA group) 15TU, which is known to be phylogenetically related to A. lwoffii and which has been reportedly isolated from sewage, freshwater aquaculture habitats, trout intestines, and frozen shrimp (6). 
 
One colony per sample was retained for further investigation (isolates BY1 to BY9). Susceptibility testing and MIC determinations were performed by disk-diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France) and Etest (AB bioMerieux, Solna, Sweden) (Table). All isolates except 1 were resistant to penicillins, combinations of penicillins and β-lactamase inhibitors, and carbapenems but susceptible to cefotaxime and of reduced susceptibility to ceftazidime. Isolate BY1 showed higher MICs for carbapenems (Table). In addition, all isolates were resistant to tetracycline, kanamycin, and fosfomycin and remained susceptible to fluoroquinolones, chloramphenicol, gentamicin, amikacin, tobramycin, and sulfonamides. Susceptibility profiles of 3 Acinetobacter genomospecies 15TU reference strains showed that they were fully susceptible to penicillins, carbapenems, tetracycline, and kanamycin. 
 
 
 
Table 
 
Antimicrobial drug MICs for Acinetobacter genomospecies 15TU isolates from cows and reference strains, France, August 2010 
 
 
 
Clonal diversity between the isolates was assessed by pulsed-field gel electrophoresis (5), which showed 6 distinct genotypes. Isolate BY1 corresponded to a single clone (data not shown), which indicated that the occurrence of Acinetobacter genomospecies 15TU strains among these animals was not the result of dissemination of a single clone. 
 
PCR detection and sequencing of genes that encode carbapenem-hydrolyzing class D β-lactamases (5) showed that the 9 Acinetobacter genomospecies 15TU isolates harbored a blaOXA-23 gene, whereas the 3 reference strains remained negative. Sequencing confirmed that all isolates expressed β-lactamase OXA-23, which is known to be widespread in A. baumannii. 
 
Mating-out assays and plasmid electroporation assays were performed by using blaOXA-23–positive Acinetobacter spp. isolates as donors and rifampin-resistant A. baumannii BM4547 isolates as a recipient strain (5); however, these assays were unsuccessful. Plasmid DNA analysis (5) gave uninterpretable results, with DNA degradations. 
 
The genetic structures surrounding the blaOXA-23 gene were investigated by PCR mapping (7), which identified transposon Tn2008 in isolate BY2 only. Tn2008 is a major vehicle for the spread of the blaOXA-23 gene in A. baumannii in the People’s Republic of China (8) and the United States (9). In the other isolates, the ISAba1 element of Tn2008 had been truncated by a novel insertion sequence termed ISAcsp2 (www-is.biotoul.fr). 
 
The dairy farmer indicated that most animals from which OXA-23 producers had been identified had received antimicrobial drugs in the previous weeks. Although 1 animal had received amoxicillin-clavulanate, most of the others had been given oxytetracycline and neomycin to treat mastitis. 
 
β-lactamase OXA-23 is a common source of carbapenem resistance in A. baumannii (5). Infections with multidrug-resistant OXA-23–producing A. baumannii or A. junii have been reported from hospitals but not from the community. Our study showed that OXA-23–producers in particular, and carbapenemase producers in general, may be isolated from animals. Among the hypotheses that could explain the selection of this carbapenemase, use of penicillins or penicillin–β-lactamase inhibitor combinations could create selective pressure for β-lactamases because OXA-23 does confer, in addition to decreased susceptibility to carbapenems, a high level of resistance to those compounds. We have previously shown that A. radioresistens, an environmental species, was the progenitor of the blaOXA-23 gene (10). Studies are needed to determine to what extent and at which locations Acinetobacter genomospecies 15TU and A. radioresistens might co-reside and therefore where the blaOXA-23 gene exchange might have occurred.

23-producing A. baumannii is considered a serious threat; those strains are frequently involved in nosocomial outbreaks for which therapeutic options are extremely limited (3,4). Our study objective was to evaluate the possible occurrence of carbapenemase-producing gramnegative bacteria in dairy cattle in France.
In August 2010, at a dairy farm 30 km from Paris, France, rectal swabs were collected from 50 cows. Samples were precultured in buffered peptone water and incubated for 18 h at 37°C. Cultures were inoculated by streaking 100 μL of the suspensions onto Drigalski agar plates (bioMérieux, Balmes-les-Grottes, France) containing 1 μg/mL of imipenem to select for carbapenem-resistant gramnegative isolates. Of the 50 samples, 9 produced growth on imipenemcontaining plates. All colonies tested (10 colonies/sample) by using the API 20 NE (bioMérieux) system were fi rst identifi ed as A. lwoffi i. Molecular techniques based on sequencing of the gyrA, gyrB, and rpoB genes (5) enabled more precise identifi cation and indicated that all isolates belonged to the Acinetobacter genomospecies (DNA group) 15TU, which is known to be phylogenetically related to A. lwoffi i and which has been reportedly isolated from sewage, freshwater aquaculture habitats, trout intestines, and frozen shrimp (6).
One colony per sample was retained for further investigation (isolates BY1 to BY9). Susceptibility testing and MIC determinations were performed by disk-diffusion assay (Sanofi -Diagnostic Pasteur, Marnesla-Coquette, France) and Etest (AB bioMérieux, Solna, Sweden) (Table). All isolates except 1 were resistant to penicillins, combinations of penicillins and β-lactamase inhibitors, and carbapenems but susceptible to cefotaxime and of reduced susceptibility to ceftazidime. Isolate BY1 showed higher MICs for carbapenems (Table). In addition, all isolates were resistant to tetracycline, kanamycin, and fosfomycin and remained susceptible to fl uoroquinolones, chloramphenicol, gentamicin, amikacin, tobramycin, and sulfonamides. Susceptibility profi les of 3 Acinetobacter genomospecies 15TU reference strains showed that they were fully susceptible to penicillins, carbapenems, tetracycline, and kanamycin.
Clonal diversity between the isolates was assessed by pulsedfi eld gel electrophoresis (5), which showed 6 distinct genotypes. Isolate BY1 corresponded to a single clone (data not shown), which indicated that the occurrence of Acinetobacter genomospecies 15TU strains among these animals was not the result of dissemination of a single clone. PCR detection and sequencing of genes that encode carbapenemhydrolyzing class D β-lactamases (5) showed that the 9 Acinetobacter genomospecies 15TU isolates harbored a bla OXA-23 gene, whereas the 3 reference strains remained negative. Sequencing confi rmed that all isolates expressed β-lactamase OXA-23, which is known to be widespread in A. baumannii.
Mating-out assays and plasmid electroporation assays were performed by using bla OXA-23 -positive Acinetobacter spp. isolates as donors and rifampin-resistant A. baumannii BM4547 isolates as a recipient strain (5); however, these assays were unsuccessful. Plasmid DNA analysis (5) gave uninterpretable results, with DNA degradations.
The genetic structures surrounding the bla OXA-23 gene were investigated by PCR mapping (7), which identifi ed transposon Tn2008 in isolate BY2 only. Tn2008 is a major vehicle for the spread of the bla OXA-23 gene in A. baumannii in the People's Republic of China (8) and the United States (9). In the other isolates, the ISAba1 element of Tn2008 had been truncated LETTERS by a novel insertion sequence termed ISAcsp2 (www-is.biotoul.fr).
The dairy farmer indicated that most animals from which OXA-23 producers had been identifi ed had received antimicrobial drugs in the previous weeks. Although 1 animal had received amoxicillin-clavulanate, most of the others had been given oxytetracycline and neomycin to treat mastitis.
β-lactamase OXA-23 is a common source of carbapenem resistance in A. baumannii (5). Infections with multidrug-resistant OXA-23-producing A. baumannii or A. junii have been reported from hospitals but not from the community. Our study showed that OXA-23-producers in particular, and carbapenemase producers in general, may be isolated from animals. Among the hypotheses that could explain the selection of this carbapenemase, use of penicillins or penicillin-βlactamase inhibitor combinations could create selective pressure for β-lactamases because OXA-23 does confer, in addition to decreased susceptibility to carbapenems, a high level of resistance to those compounds. We have previously shown that A. radioresistens, an environmental species, was the progenitor of the bla OXA-23 gene (10). Studies are needed to determine to what extent and at which locations Acinetobacter genomospecies 15TU and A. radioresistens might co-reside and therefore where the bla OXA-23 gene exchange might have occurred.

Acknowledgments
We thank A. Nemec and L. Dijkshoorn for the Acinetobacter genomospecies 15TU reference strains.  During September 2011, Ae. albopictus mosquitoes were collected from a cemetery in Cancun, which is located in the eastern part of the Yucatan Peninsula (21°8.53′Ν, 86°52.79′W) (Figure). The collection location was shaded by trees. Water in containers from which larvae were collected had an average temperature of 24.5°C and a pH of 8.5. The larval collection included ≈30 specimens of different developmental stages that were collected from vases and other artifi cial containers in the cemetery. Finding Ae. albopictus mosquitoes in Cancun was not surprising because these mosquitoes have been found in nearby Belize (9). Cancun is also a major port for ships carrying tourists and goods that originate in areas to which Ae. albopictus mosquitoes are endemic, including Florida and Texas. Nevertheless, the introduction of Ae. albopictus mosquitoes into Cancun and the high potential for establishment and spread across the Yucatan Peninsula has major public health implications.
The Yucatan Peninsula is hyperendemic for dengue, with all 4 dengue virus (DENV) serotypes circulating in this region. Should Ae. albopictus mosquitoes persist in this region, they may spread and come to play a secondary role to Ae. aegypti mosquitoes as local vectors of DENV. Ae. albopictus mosquitoes may also change local virus transmission dynamics. For example, DENV transmission may be intensifi ed in rural areas because Ae. albopictus mosquitoes are more likely than Ae. aegypti mosquitoes to be found in this setting. Ae. albopictus and Ae. aegypti mosquitoes also may differ in their potential for vertical transmission of DENV, which could