Characterization of Leptospira Interrogans Serovar Pomona Isolated from Swine in Brazil

Introduction: Leptospira interrogans swine infection is a cause of serious economic loss and a potential human health hazard. In Brazil, the most common serovars associated with swine infections are Pomona, Icterohaemorrhagie and Tarassovi. Cross-reactions among serovars and the failure of infected animals to seroconvert may complicate the interpretation of serological tests. Molecular methods with better discriminatory powers are useful tools for swine leptospirosis characterization and diagnosis. Methodology: This study evaluated nine L. interrogans isolates from the States of Sao Paulo and Minas Gerais during different time periods. Isolates from diseased and apparently healthy swine were characterized by microscopic agglutination tests with polyclonal antibodies and were genotyped by VNTR, PFGE and MLST techniques. Broth microdilution was used to determine the minimal inhibitory concentration of the antimicrobials of veterinary interest. Results: The strains were identified as L. interrogans serogroup Pomona serovar Pomona Genotype A, while MLST grouped all of the isolates in sequence type 37. The PFGE analysis resulted in two pulsotypes with more than 70% similarity, distinguishing serovar Pomona isolates from the serovar Kennewicki reference strain. All of the isolates presented low MIC values to penicillin, ampicillin, ceftiofur and tulathromycin. High MIC values for fluoroquinolones, tiamulin, gentamicin, tetracyclines, neomycin, tilmicosin and sulfas were also observed. Conclusions: All molecular techniques were concordant in L. interrogans serovar Pomona identification. This serovar may have a different antibiotic susceptibility profile than previously reported for Leptospira isolates.


Introduction
Leptospirosis is an important infectious disease, which is reemerging in some developing countries, and is a globally spread zoonosis [1].Leptospira interrogans infection in swine is a cause of serious economic losses and a potential human health hazard [2].Swine are commonly infected with serovars Pomona, Tarassovi, Grippotyphosa, Bratislava, Sejroe, Icterohaemorrhagiae and Canicola, and the clinical signs of swine leptospirosis vary with the infecting serovar [3].
In Brazil, the most common serovars described are Pomona, Icterohaemorrhagie and Tarassovi.Infection with serovar Pomona is characterized by high antibody titers, variable clinical signs and a renal carrier state [4].Icterohaemorrhagiae infection is associated with an increased risk of weak newborn piglets and stillbirths in Brazil [5]; serovar Tarassovi is associated with dead piglets [2].Seroprevalence may vary with management practices, the presence of surface water and a farm rodent-control program [6,7].Crossreactions among serovars, diagnostic titer uncertainty, and failure of some infected animals to seroconvert cause considerable difficulty in serological test interpretation [8].Thus, molecular methods with higher discriminatory capabilities are useful tools for swine leptospirosis characterization and diagnosis.
Genotyping methods, such as pulsed-field gel electrophoresis (PFGE), have been standardized for Leptospira characterization [9] in addition to simple PCR techniques, such as variable number tandemrepeat analysis (VNTR) [10].The increasing number of sequenced genomes enables the use of sequencebased approaches such as multilocus sequence typing (MLST) for concrete serovar identification [11].A microdilution technique was developed for antibiotic selection and leptospirosis treatment [12].
This study aimed to characterize Leptospira interrogans serovar Pomona isolated from swine in Sao Paulo and Minas Gerais states during different time periods by serotyping, variable number tandemrepeat analysis (VNTR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and broth microdilution techniques.

Bacterial isolates and culture conditions
Nine Leptospira interrogans isolates were studied.The isolates originated from the Laboratory of Bacterial Zoonosis -University of Sao Paulo bacterial collection.They were isolated from diseased and apparently healthy swine in Sao Paulo and Minas Gerais states at three different time periods (Table 1).

Serotyping
The isolates were typed at the WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands).For the serogroup determination, the isolates were subjected to microscopic agglutination tests (MAT) using a panel of 43 rabbit anti-Leptospira sera representative of pathogenic and saprophytic serogroups.The polyclonal rabbit antisera were prepared as previously described [15,16].The MAT was performed using a microtiter plate with serial 2-fold rabbit antisera dilutions, starting with a serum dilution of 1:10.Equal volumes of viable leptospiral strains and antisera dilutions were mixed.After incubation at 30°C for 2 hours, the mixtures were investigated for agglutination using dark-field microscopy.High agglutination rates of the leptospiral strain with one particular antisera, were used to identify the presumptive serogroup of the strain [17].

Broth microdilution
Broth microdilution was performed as previously described [12] and adapted for use with the Sensititre Standard Susceptibility MIC Plate BOPO6F (Thermo Fisher Scientific -Waltham, USA).For the inoculum, cultures were grown at 30°C for 7 days and diluted to an optical density at 420 nm of 0.32 (approximately 10 8 CFU/mL), followed by serial dilution using EMJH medium to achieve a final concentration of approximately 2×10 6 CFU/mL.Fifty microliters of the inoculum was added in each Sensititre MIC Plate well, and after 3 days of incubation, 5 μL of 10X alamarBlue (Thermo Fisher Scientific -Waltham, USA) was added to each well.The MICs were assessed visually as the lowest antibiotic concentration in the wells without alamarBlue color change at the fifth day of incubation.

Variable number tandem-repeat analysis (VNTR)
VNTR analysis was performed with 7 discriminatory markers for VNTR loci 4, 7, 9, 10, 11, 19 and 23 as previously described [10] at the Biotechnology Centre, Federal University of Pelotas, Rio Grande do Sul, Brazil.The PCR products were analyzed on a 1.5% agarose gel stained with ethidium bromide, and the molecular weights were estimated with a 100-bp DNA ladder.

Pulsed-field gel electrophoresis (PFGE)
The Leptospira isolates were incubated at 30°C in EMJH medium for 7 days and centrifuged at 5000 rpm for 20 minutes.The supernatant was discarded and the pellet was resuspended in 10 mL of PETT IV solution (10 mmol/L Tris-HCl [pH 8.0], and 1 mol/L NaCl, 10 mmol/L EDTA).The bacterial suspension was centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded, and the pellet was suspended in 1 mL of lysis buffer (1 mol/L NaCl, 10 mmol/L Tris [pH 8.0], 200 mmol/L EDTA, 0.5 % sarcosyl, and 0.2 % sodium deoxycholate).Agarose SeaKem Gold 2% (Lonza Group Ltd -Basel, Switzerland) was prepared in 0.5X Tris Borate EDTA buffer.A 400 μL bacterial suspension aliquot was heated to 40°C and was added to 20 μL of 100 mg of lysozyme/mL (LGC Biotecnologia, Sao Paulo, Brazil) and 400 μL of a heated 2% agarose solution.The mixture was immediately dispensed into wells and chilled for 10 minutes at 4°C.Plugs were placed in 2.5 mL of lysis buffer, and 70 μL of proteinase K (20 mg/ml; LGC Biotecnologia) were added before incubation at 56°C for 20 hours.The plugs were rinsed once in 1 mL of Tris EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA).The plugs were washed with 5 mL of Tris EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA) twice for 30 minutes, each time at 37°C, and then stored in 1 mL of Tris EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA) at 4°C.The DNA was cleaved with 5 U of NotI enzyme (New England BioLabs, Ipswich, MA, USA) per microgram of DNA for 4 hours at 37°C.PFGE was performed as previously described [9].The gels were stained with 1x Sybr Safe (Invitrogen Corporation, Carlsbad, USA) for 40 minutes and photographed with a Gel Doc XR System (Bio-Rad Laboratories, Hercules, USA).The DNA fragments were identified using a Lambda DNA-PFGE marker (New England BioLabs, Ipswich, USA).For the PFGE analysis, the isolates were categorized into different pulsotypes when differing by 4 or more bands [18].Fingerprint patterns were analyzed by comprehensive pairwise comparison of restriction fragment sizes using the Dice coefficient.The mean values obtained from Dice coefficients were employed in UPGMA using BioNumeric 6.6 (Applied Maths NV, Saint-Martens-Latem, Belgium) to generate dendrograms.
Amplified fragments were purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare do Brasil Ltda, São Paulo, Brasil) and sequenced directly by the Human Genome Research Center (University of Sao Paulo, Brazil).The BIOEDIT Sequence Alignment Editor 7.0.9[21] was used for sequence editing.Sequences were submitted to the Leptospira MLST database (http://www.mlst.net) to obtain isolates allelic profiles and respective sequence types (ST).

Nucleotide sequence accession numbers
All DNA sequences from this study were deposited in GenBank under accession numbers KJ885311 to KJ885373.
PFGE analysis resulted in two pulsotypes (P1 and P2).The pulsotype P1 grouped all of the studied isolates, including the serovar Pomona reference strain 13A, with more than 90% genetic similarity; P2 only represented the reference strain LPF (Figure 1).The serovars Pomona and Kennewicki corresponding to the pulsotypes P1 and P2, respectively, had over 70% genetic similarity.All of the isolates presented the same allelic profile (3, 3, 3, 2, 3, 4, 5) and sequence type (ST 37) obtained from the MLST analysis.
The MIC values are presented in Table 2.All of the isolates had high MIC values to tiamulin, gentamicin, chlortetracyclin, oxytetraciclin, neomycin, tilmicosin, trimethoprim/sulfamethoxazole, spectinomycin, sulfadimethoxine.High fluoroquinolone MIC values were also observed.All of the isolates appear to be sensitive to penicillin, ampicillin, ceftiofur, tylosin tartrate and tulathromycin.MIC variability was observed for florfenicol.Isolate GR5 presented a slightly different profile with low MIC values for gentamicin, florfenicol, chlortetracyclin and oxytetracycline, while it appeared to be resistant to clindamycin.

Discussion
We characterized L. interrogans serovar Pomona isolates from diseased and apparently healthy swine.Despite the distinct sample types, origin and three different isolation time periods, the studied isolates presented an identical genotype and ST and a similar antibiotic susceptibility profile.
The serotyping presents the same result as the molecular techniques, with the identification of serovar Pomona; however, molecular approaches gave us more information identifying genotype A strains, which differs from the L. interrogans serovar Pomona reference strain.Furthermore, serological methods still are laborious, especially for antisera production that involves animal use [8].
Regarding molecular typing methods in this study, VNTR analysis allowed us to identify the serovar Pomona genotype A and clustered the isolates into a single group.This genotype was first isolated from Argentinian cow, pig and human samples [22].Genotype A is dominant among the Argentinian territory and has been isolated since 1960 with a 55% prevalence [23].Despite not fully understanding the epidemiological importance of this genotype, we can conclude that it is present in South America and that it can be isolated from both healthy and diseased animals.
The PFGE technique enabled the isolates differentiation in only two pulsotypes.The field isolates presented over 90% similarity with reference strain 13A and grouped in pulsotype P1, while P2 represented only the serovar Kennewicki reference strain.Therefore, PFGE enabled the distinction of serovars Pomona and Kennewicki with more than 70% genetic similarity.Although serovar Kennewicki is no longer separated from serovar Pomona [24], PFGE is a useful method for serovar identification, as previously reported [9].
While PFGE distinguished closely related serovars, the MLST technique grouped all of the isolates into one ST.ST 37 was expected for the Pomona isolates because it had already been assigned this serovar, independently of its origin [19,9,11].As MLST does not always specify Leptospira serovars [9], the same ST 37 also encompasses serovar Canicola when using the scheme previously proposed [19,9,11].This is also observed with the new MLST protocol [25] in which serovar Pomona can be assigned to STs 38 and 140 together with serovars Gem and Guaratuba, respectively.Although MLST techniques enable Leptospira species differentiation [9,25], serovar assignment still needs improvement.Only when molecular methods ensure species and serovar identification, will they completely replace serological methods.
Although a standardized Leptospira antibiotic susceptibility guideline has not been established by the Clinical and Laboratory Standards Institute, Leptospira susceptibility studies have been developed to enhance leptospirosis treatments [26].However, few field isolates and reference strains have been studied to generate an understanding of the variability of susceptibilities among Leptospira species and serovars.L. interrogans is the most studied species, but there are no data regarding the variability of L. interrogans serovars antibiotic susceptibilities.
Our results corroborate previous studies that presented high MIC values for tetracycline and chloramphenicol-florfenicol [27,12,28,29].All of the isolates were also resistant to trimethoprim/sulfamethoxazole and sulfadimethoxine, as expected.However, the higher MIC values for tiamulin, gentamicin, neomycin and spectomycin were not expected and the veterinary usage of these antibiotics requires attention.Regardless of the susceptibility to penicillin and ampicillin that consists of a classical leptospirosis treatment, all serovar Pomona isolates appear to be resistant to fluoroquinolones, which have been indicated as an alternative empirical treatment [26].
These results indicate that L. interrogans serovar Pomona genotype A has a different antibiotic susceptibility profile compared to other L. interrogans serovar [27,28,29,30] and although they are sensitive to penicillin and ampicillin, they also have high MIC values for important veterinary medicine antibiotics, such as tetracycline, chloramphenicol-florfenicol, aminoglycosides and tiamulin.Fluoroquinolone (danofloxacin and enrofloxacin) resistance in Brazilian leptospires has been reported by Miraglia et al. [30] and contradicts previous publications from other research laboratories.

Conclusions
Molecular techniques were concordant in L. interrogans serovar Pomona identification.This serovar presents a different antibiotic susceptibility profile compared to other Leptospira interrogans strains previously reported.Further studies with large numbers of strains are necessary to define the susceptibility profile of serovar Pomona.

a
The MICs of penicillin are given in units/mL. 1L. interrogans serogroup Pomona serovar Pomona; 2 L. interrogans serogroup Pomona serovar Kennewicki strain Fromm.

Figure 1 .
Figure 1.A dendrogram showing the relationship among the L. interrogans serogroup Pomona isolates PFGE patterns.

Table 1
Source and origin characteristics and molecular profile of L. interrogans serogroup Pomona isolates

Table 2
Minimal inhibitory concentration (MIC) values of antimicrobials against L. interrogans serogroup Pomona isolates