Identification of novel OXA-134-like β-lactamases in Acinetobacter lwoffii and Acinetobacter schindleri isolated from chicken litter

Background. Various food-producing animals have been recognized in recent years as a potential reservoir for the spread of antibiotic resistant bacteria that may pose a risk to human health and therefore their dissemination in the food production chain needs to be assessed. Methods. In this study, 450 boot swabs from chicken farms were analyzed for the presence of antimicrobial resistance with a focus on β-lactams resistance in Acinetobacter species. Results. Two β-lactamase-encoding genes were first time identified in Acinetobacter lwoffii and Acinetobacter schindleri isolates. The deduced amino acid sequence of OXA-496 shared 93.8% identity with OXA-363. The second OXA-134-like enzyme, OXA-537, had the highest sequence identity (97.8%) with OXA-235 and OXA-237. Conclusions. The results of this study illustrate the occurrence of new OXA-134-like β-lactamases, called OXA-496 and OXA-537, carrying strains of Acinetobacter lwoffii and Acinetobacter schindleri in chicken farm litter, and highlight the possible role of Acinetobacter as a reservoir of resistance genes.

Nosocomial infections caused by members of the genus Acinetobacter have been most often attributed to A. baumannii.However rarer, infections mainly associated with catheter-related bloodstream infections are caused by A. lwoffii and Acinetobacter schindleri [14][15][16] .Moreover, there is a growing concern over the transmission of ESBL and/or carbapenemase-producing Acinetobacter spp. in food producing animals (cattle, pig, horse and chicken) in many countries in Europe and Asia 2,4-6 .Therefore, the role of healthy farm animals as a potential reservoir for the spread of antibiotic resistant bacteria and their impact on the food processing chain has to be assessed.
The aim of the current study was to investigate for the occurrence of β-lactams resistance in Acientobacter species from chicken farm litter.

Bacterial isolation
From November 2014 to March 2015, 450 boot swabs of farm staff were analyzed from chicken farms in Moravia (Czech Republic).Boot swabs were suspended in peptone water (Trios) and were grown aerobically at 37 °C for 24 h.A loopful of peptone water was streaked on Brilliance TM CRE agar plates (Oxoid).The agar plates were incubated for 24 h aerobically at 37 °C.The isolates that grew were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) (Bruker Daltonik GmbH, Bremen, Germany) and were further tested for antibiotic susceptibility.
Twenty samples were taken from the 1) bedding material, 2) feed, 3) scrapings from the floor and 4) water before the poultry were placed and microbiologically examined in order to exclude microbial contamination in the chicken farm.Culture tests focused specifically on detection of Acinetobacter spp.Samples from the bedding material (1) and feed (2) were suspended in peptone water (Trios) and were grown aerobically at 37 °C for 24 h.A loopful of peptone water was streaked on Endo agar (Trios) and Brilliance TM CRE agar plates (Oxoid).The agar plates were incubated for 24 h aerobically at 37 °C.Disinfection control of hall floor prior to the housing of poultry was carried out by scraping the surface (100 cm 2 ) using sterile swabs moistened with peptone water (3).The swab after delivery to the laboratory was incubated for 24 h in peptone water at 37 °C followed by inoculation of blood agar (Trios) and Endo agar (Trios), which were again incubated aerobically at 37 °C overnight.Water samples (4) for the detection of Acinetobacter spp.were examined by the modified method according to CSN EN/ISO 16266 (ref. 17).This consisted of adding Endo agar (Trios) to the above mentioned solid culture media (Trios).Identification of isolates was performed in all cases after subculturing on blood agar by MALDI.

Design of primers for PCR amplification
The sequences of genes encoding β-lactamases and carbapenemases (bla KPC , bla VIM , bla IMP , bla NDM , bla OXA ) so far described (http://bldb.eu;last accessed October 2017) (ref. 19) were downloaded from the GenBank databases.Alignment was performed using Geneious Pro 8.1.9(Biomatters, New Zealand) to identify homologous regions.Primers were then designed using Geneious software.In addition, a pair of flanking primers described by Kamolvit et al. 20 was used to amplify and sequence the entire bla OXA gene.Detailed primer specifications are given in Table 2.The isolates were tested by PCR for the presence of β-lactamase and carbapenemase genes: bla KPC , bla VIM , bla IMP , bla NDM and bla OXA 21-23   .Expression levels of the bla OXA genes were not investigated.

DNA extraction and amplification conditions
Genomic DNA isolation was similar as described by Mlynarcik et al. 23 .The PCR reaction mixture contained: 1 µL DNA (100 ng) in 24 µL complete reaction buffer  2.

Identification of Acinetobacter species
Acinetobacter isolates were identified by MALDI and partial rpoB gene sequencing as described elsewhere 24 .Sequences of rpoB gene (seqtypes) were included for analysis.Primers used for PCR amplification and sequencing are listed in Table 1.A neighbor-joining tree was constructed using the Geneious Tree Builder (Geneious Pro 8.1.9,Biomatters, New Zealand) using Tamura-Nei Algorithm with 1000 bootstrap samplings.The rpoB gene sequence of Pseudomonas aeruginosa PAO1 (AE004091) was used as an outgroup.In comparative studies, sequences of A. schindleri strain LUH 5677 (KU961630), A. schindleri strain LUH 7428 (KU961631), A. lwoffii strain LMG 1300 (EF611398) and A. lwoffii strain LUH 1710 (KU961624) were also included.

Epidemiological setting
Poultry litter in the halls was composed of thick wood shavings that normally do not come into contact with the soil, surface water or wild living animals.So if new litter material is brought into the hall, it is subjected to bacteriological investigation for any pathogenic bacteria.In order to prevent the entry of any potential disease and to reduce the stress of poultry, the movement of people in the hall is minimal.Feeding and water supply (municipal water) are controlled by automatic machines and they are regularly monitored for bacteriological contamination.The halls are protected against flying insects, wild birds and rodents by a biosecurity program.Workers and visitors are required to walk through disinfectant mats and must wear a clean lab coat and shoe covers.Biosecurity measures are strictly followed by farmers in order to reduce the risk of infections, especially avian influenza, Newcastle disease, and salmonellosis.The halls are mechanically cleaned and then disinfected after poultry leave the farm.The breeding hall is restocked only after satisfactory disinfection as determined from microbiological testing of swab samples from the floor.

RESULTS
A total of 55 Gram-negative bacilli were obtained from selective agar plates, from which 2 (3.6%), 3 (5.5%), 4 (7.3%), 6 (10.9%) and 9 (16.4%)isolates were identified as A. radioresistens, Acinetobacter pittii, Acinetobacter calcoaceticus, A. lwoffii and A. schindleri, respectively.Isolates of Acinetobacter species from the bedding material of chickens were subjected to susceptibility testing and screening for β-lactamase and carbapenemase production.However, the isolates exhibited high susceptibility to specific β-lactam antibiotics and the presence of studied resistance genes (bla KPC , bla VIM , bla IMP and bla NDM ) has not been confirmed (data not shown).Further, the ESBL and AmpC β-lactamases were not detected in either strain.Interestingly, two Acinetobacter isolates were positive by  using OXA-5F/R primers for detection of bla OXA-235 to -237/- 278/-360 genes (See Table 2 for primer sets).Sequence analysis of the amplicon obtained from A. lwoffii isolate S459, generated with primers OXA-7F and OXA-7R (ref. 20), revealed the presence of a new member of the large OXA family and showed the highest amino acid identity to OXA-363 at 93.8% (Fig. 1) and was named OXA-496.In silico analysis revealed that the sequence located upstream (159 bp segment) had no insertion sequence element (https://www-is.biotoul.fr//)(data not shown).Sequence analysis of the amplicon obtained from A. schindleri isolate S35 showed the presence of a new variant of the OXA family, with the highest amino acid identity to OXA-134 family carbapenem hydrolyzing class D β-lactamase OXA-235 and OXA-237 at 97.8% (Fig. 1) and was named OXA-537.However, based on short sequence length of the up-and down-stream regions of the bla OXA gene we were not able to predict an insertion sequence element.
Using the broth microdilution method, A. lwoffii isolate S459, was resistant to cotrimoxazole and tetracycline, had intermediate resistance to ciprofloxacin, and maintained susceptibility to ampicillin, ampicillin/sulbactam, cefepime, meropenem, ceftazidime and piperacillin (Table 1), based on the currently available breakpoints 18 .Another interesting isolate, A. schindleri isolate S35, was intermediately resistant to tetracycline, but maintained susceptibility to meropenem, cefepime, ampicillin, ampicillin/sulbactam, ceftazidime and piperacillin (Table 1).We determined the MICs of imipenem (0.125 µg/mL) by using the E-test method which showed that the isolates were susceptible to carbapenem (Table 1).
Two Acinetobacter isolates from chicken bedding litter (S459 -seqtype 1 and S35 -seqtype 2) based on sequence analysis of zone 2 of the rpoB gene showed between 99.2%/99.1% and 99.8%/99.6%nucleotide identity to Acinetobacter type strains in GenBank, and were identified as: A. lwoffii 'close to LMG 1300 and/or LUH 1710' and A. schindleri 'close to LUH 5677 and/or LUH 7428', respectively.However, seqtype 1 showed different 3-bp or 4-bp sequence of partial rpoB gene with A. lwoffii strains LMG 1300 or LUH 1710, respectively (data not shown).The remaining seqtype 2 had only a 1-bp or 2-bp sequence difference between the rpoB genes of A. schindleri strains LUH 5677 or LUH 7428 (data not shown).The Acinetobacter isolates were further divided into two subgroups based on phylogenetic clustering (Fig. 2).
To exclude microbial contamination within the chicken farm, twenty samples were taken from the bedding material, feed, scrapings from the floor and water before the poultry were placed but no Acinetobacters were isolated in any of the samples.

DISCUSSION
All isolates of Acinetobacter species were subjected to susceptibility testing and screening of β-lactamase and carbapenemase production.Isolates showed high sensitivity to specific β-lactam antibiotics and did not confirm the presence of the resistance genes examined.However, two isolates of Acinetobacter (A. lwoffii -S459 and A. schindleri -S35) from the chicken bedding litter were positive for two novel bla OXA-134-like genes, bla OXA-496 and bla OXA-537 .These results can be explained by the absence of insertion sequence in upstream position of bla OXA-like genes which provides the strong promoter sequence.It is therefore likely that bla OXA-134-like genes were not expressed (or expressed at negligible level) in these hosts.In this study, the expression of the bla OXA-134-like genes was not studied.Moreover, it has been described that the OXA-134-like enzymes are intrinsically present in A. lwoffii and A. schindleri 25,26 .This has therefore proved to be a useful means of identification of these species.Although it could be a potential reservoir for antibiotic resistance.This suggests that our two new genes encoding OXA-134-like enzymes can be considered as intrinsic and unique to A. lwoffii and A. schindleri, but this has not been the subject of our study.
In this study, partial rpoB gene sequencing analysis revealed two isolates (S459 -seqtype 1 and S35 -seqtype 2) were A. lwoffii 'close to LMG 1300' and A. schindleri 'close to LUH 5677', respectively.Further, it has been reported that the A. lwoffii LMG 1300 can be isolated from soil and human clinical samples 15,27 .However, A. schindleri LUH 5677 has been identified as animal-associated (isolated from trachea python) (ref. 28).Routine farm maintenance prevents poultry placement until halls are deemed microbial free.To support our findings twenty samples were taken from the bedding material, feed, scrapings from the floor and water before the poultry were placed and were sent to the microbiology laboratory for routine culture identification.However, in none of the samples were Acinetobacters isolated.These facts together lead us to believe that the strains of Acinetobacter could come from poultry but neither the environment nor human is excluded as a source of bacteria, as it is assumed impossible to have a chicken barn without microbes.
Acinetobacter are common, wild saprophytes found in soil, water, sewage and food of animal origin.A. lwoffii is one of the most prevalent Acinetobacter species involved in the economically important degradation of foods such as chicken, bacon, eggs, and fish, even when stored under cold conditions or after irradiation 29 .However, there are many other pathogenic bacteria (e.g., Pseudomonas spp.) that cause degradation of food, particularly chicken meat.In the past, Acinetobacter species were considered saprophytes of little clinical significance.However, with the introduction of new potent antibiotics into clinical practice and agriculture, infections caused by resistant Acinetobacter species occur with increasing frequency 10,13 .Thus, the identification of Acinetobacter species that carry different β-lactamases deserves continued attention.
The current epidemiology of OXA-producing bacteria in veterinary medicine is not yet fully understood, as data on the presence and distribution of these genes are lacking and therefore continuous monitoring is essential.

CONCLUSION
This is the first report describing novel OXA-134-like β-lactamases, OXA-496 and OXA-537, in A. lwoffii and A. schindleri isolated from chicken farm litter, and highlight the reservoir of resistance.Different emerging antimicrobial resistance properties in bacteria of food animal origin have been seen in the last years and further monitoring of the presence of resistance genes in these bacteria is warranted.

Fig. 2 .
Fig. 2. Phylogenetic relationships between the two isolates (seqtype 1 -Acinetobacter lwoffii isolate S459 and seqtype 2 -Acinetobacter schinderi isolate S35) and other members of the genus Acinetobacter inferred from partial rpoB DNA sequences.Numbers at nodes represent bootstrap values out of 1000 resamplings that were greater than 50%.The tree was outgroup rooted with Pseudomonas aeruginosa strain PA01 sequence.

Table 1 .
Antimicrobial susceptibilities of the two Acinetobacter isolates.
a Susceptibility categories are given in parentheses: I, intermediate; S, susceptible; R, resistant; ND, not defined.

Table 2 .
Sequences of primers used for PCR for detection of genes encoding β-lactamases and carbapenemases, and rpoB gene analysis.
a For degenerate primers: R = A or G; S = G or C; Y = C or T. b Concentration of each of the primers was 25 pmol.