Comparative Immunology, Microbiology and Infectious Diseases 38 (2015) 33–39
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Virulence gene profiling and antibiotic resistance pattern of Indian isolates of Pasteurella multocida of small ruminant origin Laxmi N. Sarangi ∗ , P. Thomas, S.K. Gupta, A. Priyadarshini, S. Kumar, Viswas Konasagara Nagaleekar, A. Kumar, Vijendra P. Singh Division of Veterinary Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, UP, India
a r t i c l e
i n f o
Article history: Received 1 January 2014 Received in revised form 11 November 2014 Accepted 20 November 2014 Keywords: Pasteurella multocida Virulence genes Small ruminants toxA pfhA Antibiogram
a b s t r a c t Pasteurellosis in small ruminants affects the livelihood of small and marginal farmers of India. The present study was undertaken to understand the trends in gene carriage and antibiotic resistance pattern of Pasteurella multocida isolates recovered from small ruminants over a period of 10 years in India. A total of 88 P. multocida isolates of small ruminant origin were subjected to virulence gene profiling for 19 genes by PCR and antibiogram study employing 17 different antibiotics. Virulence genes like exbB, exbD, tonB, oma87, sodA, sodC, nanB and plpB (100% prevalence) and ptfA and hsf-2 (>90% prevalence) were found to be uniformly distributed among isolates. Unexpectedly, a very high prevalence (95.45%) of pfhA gene was observed in the present study. Dermonecrotoxin gene (toxA) was observed in 48.9% of isolates with highest occurrence among serotype A isolates and interestingly, one of each isolate of serotype B and F were found to carry this gene. Antimicrobial susceptibility testing revealed 17.04% isolates to be multidrug resistant. Amongst all the antibiotics tested, most of the P. multocida isolates were found to be susceptible to enrofloxacin and chloramphenicol. This study highlights novel epidemiological information on frequency and occurrence of virulence genes among Indian isolates from small ruminants. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Pasteurella multocida belonging to the family Pasteurellaceae is associated with a number of economically important diseases like haemorrhagic septicaemia in cattle and buffalo; enzootic bronchopneumonia in cattle, sheep and goats; atrophic rhinitis in swine; fowl cholera in poultry and snuffles in rabbits [1,2]. In addition, this bacterium has also been isolated from various cases in domestic as well as wild animals [3].
∗ Corresponding author. Tel.: +91 9756597591. E-mail address: [email protected] (L.N. Sarangi). http://dx.doi.org/10.1016/j.cimid.2014.11.003 0147-9571/© 2014 Elsevier Ltd. All rights reserved.
P. multocida has been classified into five types (A, B, D, E and F) based on capsular typing and most often each capsule type has been found to be predominantly associated with a specific disease in a host species [4]. However, over the years, there are reports of occurrence of uncommon capsular type from different host species (reviewed in [2]), which is either due to spontaneous change from one capsular type to another or due to cross species infections [5,6]. Such an ability of the bacteria to colonize, survive and subsequently infect multiple hosts is a great concern, which necessitates a detailed study on isolates from different host origin. In India, animal husbandry practices like cohabitation of various species of animals including poultry and use of common grazing ground may lead to spread of infection among all such host species. This results in a
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complex scenario of circulation of various bacterial strains among members of host populations, notwithstanding an increased possibility of isolates jumping species barriers. Though antibiotics have been used traditionally for the treatment of pasteurellosis, their prolonged and indiscriminate use has led to onset of resistance among various strains [7–9], thus eroding the antibiotic armamentarium and limiting therapeutic options [10]. Most of the antibiotic resistance genes are present on bacterial plasmids, integrons and transposons, which lead to the spread of these genes among isolates [10]. Antimicrobial resistance of Pasteurella isolates varies according to host origin, time of infection, geographical location, anti-bacterial pretreatment and on accessibility of the isolates to the resistance genes present in the gene pool [10]. Hence, it was necessary to carry out a detailed study of the antibiotic susceptibility pattern of P. multocida isolates. Virulence profiling has been used as a typing method for characterization of bacterial pathogens [11] including P. multocida [1,9,12]. However, studies regarding the population structure and virulence gene patterns of isolates from small ruminants are scanty [1,13,14]. Therefore, the present investigation was undertaken to study the population characteristics of P. multocida isolates obtained from small ruminants by determining their virulence-associated gene profiles and phenotypic antibiotic resistance pattern.
2. Materials and methods 2.1. Bacterial isolates Eighty eight (88) P. multocida isolates recovered from small ruminants (sheep, n = 67 and goat, n = 21), maintained at Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar were used in this study. The isolates used in the study were categorized by host species origin, serotyping, year and place of origin. If more than five isolates were obtained from a particular farm/place, then only two representative isolates were selected to be included in the study. The location of the farms (districtwise) and the clinical status of the animal (when available) have been incorporated in the supplementary table.
primers used in the PM-PCR and capsular PCR are given in Table 1. 2.4. Detection of virulence associated genes by PCR All the isolates were screened for carriage of 19 virulence associated genes by uniplex PCR. The PCR was performed with 1 l of DNA, 2.5 l of 10× PCR buffer (containing 20 mM MgCl2 ), 0.5 l of 25 mM MgCl2 , 0.5 l of 10 mM dNTP mix, 1 l each of 10 pM primers, 0.25 l of 5 U Dream Taq DNA polymerase (Fermentas) and the final volume was made up to 25 l using nuclease free water. PCR mixture without DNA was used as negative control. The PCR products were analyzed in 1% agarose gel along with 100 bp DNA ladder (Gene Ruler–Fermentas) by staining with ethidium bromide. The amplified product was visualized under UV light and documented by gel documentation system. The details of the virulence genes and sequences of the oligonucleotide primers are listed in Table 1. 2.5. Antibiotic susceptibility Each of the above isolates were tested for susceptibility against 17 different antibiotics viz. ampicillin (A), amoxycillin (AM), cephalexin (CN), cefotaxime (CE), ceftriaxone (CTR), cephalothin (CH), erythromycin (E), amikacin (AK), kanamycin (K), tetracycline (T), gentamicin (G), ofloxacin (OF), pefloxacin (PF), ciprofloxacin (CF), enrofloxacin (EX), chloramphenicol (C) and co-trimoxazole [sulfamethoxazole with trimethoprim] (CO) using disc diffusion method [19]. These antimicrobials were recommended in OIE terrestrial manual chapter on haemorrhagic septicaemia and/or used by field veterinarians in different parts of India and have proven their clinical efficacy [20–22]. The test was performed by disc diffusion method recommended by Clinical and Laboratory Standards Institute (CLSI; formerly the NCCLS) and the interpretations were carried out as per CLSI standards (Performance Standard for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals M31-A2) [23]. The ranges for susceptible, intermediate and resistant for each drug used in this study are given in Table 2. 2.6. Statistical analysis
2.2. Culture and biochemical tests The isolates were revived by 18–24 h incubation in brain heart infusion (BHI) broth at 37 ◦ C and plated subsequently onto blood agar to study cultural characteristics. They were then tested for purity (biochemical tests: indole, citrate, MR, VP and sugar fermentation tests) as per standard techniques [15].
2.3. Molecular confirmation of P. multocida by P. multocida specific PCR (PM-PCR) and capsular typing The genomic DNA was isolated by CTAB method [16]. The isolates were reconfirmed as P. multocida by PMPCR [17] followed by capsular typing using the primers employed by Townsend et al. [18]. The sequences of the
Statistical analysis of the data generated from the study was performed with SPSS 16.0 (SPSS Inc., Chicago). P values of 90% prevalence). Dermonecrotoxin gene was observed in 48.9% of isolates with highest prevalence among serotype A isolates followed by capsular type D. We observed this gene in one isolate each of serotype B and F (Table 3; Supplementary Table).
In the present study, P. multocida isolates, recovered from small ruminants over a period of years in India, were tested for virulence gene carriage and antibiotic resistance patterns. With the sole exception of capsular type E, all other capsular types have been isolated from small ruminants with majority of the strains from capsular type A followed by B, D and F respectively (Data not shown). Capsular type A strains have been implicated in causing
100 100 100 100 100 93.2 100 50 100 80 54.2 41.7 16.7 83.3 40 64.4 8.3 50 16.7 0 50.8 25.0 50.0 16.7 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 52.5 75.0 50.0 66.7 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 83.1 83.3 100 66.7 40 50.8 58.3 66.7 50.0 60 66.1 91.7 100 83.3 60 94.9 96.6 100 100 100 100 100 100 80 60
69.5 66.7 83.3 66.7 20
89.5 95.2 90.9 49.2 57.1 51.1 53.7 33.3 48.9 49.2 23.8 43.2 100 100 100 100 100 100 100 100 100 100 100 100 56.7 47.6 54.5 100 100 100 100 100 100 100 100 100 83.6 71.4 80.7 49.2 66.7 53.4 73.1 47.6 67.0 73.1 71.4 72.7 95.5 95.2 95.5
NanB NanH Exb D Tonne B Exb B TbpA HasR Positive strains percentage (%)
4. Discussion
No of strains
In this piece of investigation, 35 isolates (39.77%) were found to be susceptible to all the antibiotics under study. Majority of isolates (94%) were susceptible to enrofloxacin and chloramphenicol (Table 4). Species-wise susceptibility pattern suggested that enrofloxacin and chloramphenicol had most of the ovine isolates susceptible to them, followed by tetracycline, gentamicin, ciprofloxacin, ofloxacin and co-trimoxazole. Similarly, most of the caprine isolates were susceptible to enrofloxacin, followed by chloramphenicol, gentamicin, kanamycin, cephalothin, ofloxacin, tetracycline and co-trimoxazole. Among all the antibiotics studied, resistance against ampicillin, amoxycillin and erythromycin were observed in many isolates (Table 4).
Origin
3.3. Antibiogram study
Table 3 Prevalence of virulence associated genes in Pasteurella multocida in respect to host and capsular type.
Chi-square and Fisher’s exact test analysis of association of genes among P. multocida isolates indicated a significant association between pfhA-ptfA, pfhA-hgbB, pfhA-hasR, ptfAhgbB, ptfA-nanH, hgbA-hgbB, hgbA-hasR, hgbA-tbpA, hgbAhsf-1, hgbB-tbpA, hgbB-nanH, hgbB-plpE, tbpA-nanH, tbpAtoxA, plpE-toxA and hsf-1-hsf-2 genes.
SodA
Source: Clinical and Laboratory Standards Institute (CLSI). (Performance Standard for Antimicrobial Disc and Dilution Susceptibility Test for Bacteria Isolated from Animals M31-A2). The interpretation criteria for veterinary pathogens were used. For cephalexin, the interpretation criterion for cefazolin was used as both the antibiotics belonged to same class of antibiotics. Similarly, for cefotaxim and ceftriaxone the criterion for ceftiofur was used and for ofloxacin, pefloxacin and ciprofloxacin the interpretation criterion for enrofloxacin was used. For co-trimoxazole, the interpretation criterion for potentiated sulfonamides (trimethoprimsulfamethoxazole) was used.
95.5 95.2 95.5
Hsf-2 Hsf-1 Tox A
13 13 14 17 17 14 13 14 13 14 12 16 16 16 16 12 10
Plp E
14–16 14–17 15–17 18–20 18–20 15–17 14–22 15–16 14–17 15–18 13–14 17–20 17–20 17–20 17–20 13–17 11–15
Plp B
17 18 18 21 21 18 23 17 18 19 15 21 21 21 21 18 16
Sod C
Ampicillin Amoxycillin Cephalexin Cefotaxime Ceftriaxone Cephalothin Erythromycin Amikacin Kanamycin Tetracyline Gentamicin Ofloxacin Pefloxacin Ciprofloxain Enrofloxacin Chloramphenicol Co-trimoxazole
HgbB
Resistant (≤)
HgbA
Intermediately susceptible
Ptf A
Susceptible (≥)
PfhA
Antibacterial agent
Oma 87
Table 2 Zone diameter interpretation chart (inhibition zone diameter in mm).
100 100 100
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Host origin 67 Sheep 21 Goat Total 88 Capsule type A 59 12 B 6 D 6 F 5 NT
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Table 4 Antibiotic susceptibility test result of the Pasteurella multocida isolates in respect to host organism. Host organism
Antibiotic Susceptibility
Sheep (n = 67)
S I R S I R S
Goat (n = 21)
Total (sheep and goat) (n = 88)
I R
Positive strains percentage (%) to individual antibiotics
EX
CN
K
AK
A
AM
CE
E
CTR
CF
PF
T
CO
OF
C
G
CH
94.0 6.0 0 95.2 4.8 0 94.3
70.1 15.0 14.9 81.0 14.3 4.7 72.7
79.1 13.4 7.5 90.5 4.7 4.7 81.8
80.6 14.9 4.5 71.4 23.8 4.8 78.5
34.3 28.7 37.3 42.8 14.3 42.8 36.4
59.7 16.1 23.9 71.4 9.5 19.0 62.5
50.8 35.8 13.4 42.9 52.4 4.7 48.9
28.9 53.7 19.4 23.8 71.4 4.8 26.2
74.6 20.9 4.5 66.7 28.6 4.7 72.7
82.1 13.4 4.5 76.2 19.0 4.8 80.7
74.6 17.9 7.9 66.7 28.6 4.7 77.2
89.5 3.00 7.5 80.9 14.8 4.8 87.5
82.1 8.9 8.9 80.9 4.7 14.8 81.9
82.1 14.9 3.0 85.7 9.5 4.8 82.9
94.0 6.0 0 95.2 4.8 0 94.3
86.6 10.4 3.0 90.5 9.5 0 79.5
77.6 14.9 7.5 85.7 4.8 9.5 79.5
5.7 14.8 0 12.5
11.4 6.8
17.0 4.5
25.0 38.6
14.8 22.7
39.8 11.3
57.9 15.9
22.7 4.5
14.8 4.5
15.9 6.8
5.7 6.8
7.9 10.2
13.6 3.4
5.7 10.2 0 2.3
12.5 8.0
(H = susceptible; I = intermediately susceptible; R = resistant; EX = enrofloxacin; CN = cephalexin; K = kanamycin; AK = amikacin; A = ampicillin; AM = amoxycillin; CE = cefotaxime; E = erythromycin; CTR = ceftriaxone; CF = ciprofloxacin; PF = pefloxacin; T = tetracycline; CO = co-trimoxazole; OF = ofloxacin; C = chloramphenicol; G = gentamicin; CH = cephalothin).
pneumonia in small ruminants [4], but the precise role of other capsular types in disease process is still not clear. Although there are a few reports regarding isolation of serogroup F strains from sheep suffering from respiratory and allied symptoms [24,25], still there is a need for careful analysis of pathogenic potential of these strains [1]. Productive infection by bacterial pathogens relies on the expression of various proteins that have wide ranging functions like competence, adherence, capsule synthesis and evading host immune responses. These proteins or virulence factors are involved in the disease process and are subjected to different selection pressure leading to varying degrees of inter-strain heterogeneity which can be used to assess intra-species diversity and to establish epidemiological relationships [6]. Almost all established virulence associated genes were screened in the present study to obtain comprehensive data on the prevalence of these genes in small ruminant isolates of Indian origin. Genes like ompA, ompH, psl, etc. were not included in this study as previous studies have reported 100% prevalence [1,9]. In P. multocida, different receptors like transferrin receptors (TbpA), heme receptors (HgbA, HgbB, HasR) and Ton B complex (TonB, ExbB, ExbD) are involved in adapting to variations in the supply of different heme iron sources (reviewed in [26]). Among these, TbpA protein has been reported to be an important virulence factor and epidemiological marker in cattle [1,27]. In our study, a high frequency (80.68%) of tbpA was observed, suggesting significant association of this gene with small ruminant isolates. Between two proteins (HgbA and HgbB) involved in acquiring iron directly from the heme component, gene hgbA is present in almost all isolates, whereas prevalence of hgbB varies among strains of different host origin and also with disease status of the animal [1,28]. In contrast to the finding of Shayegh et al. [13] on the low prevalence of hgbB gene among sheep isolates, our study revealed a higher prevalence (67%) of this gene. The frequency of hgbB gene was lower among goat isolates than sheep isolates and this difference between them was found to be statistically significant (p < 0.05). However, due to lack of availability of proper history of majority of the isolates with respect to disease status, it was not possible to perform association
study between virulence gene and disease status of the animal. The basic prerequisite for bacterial infections is its attachment to host cell and therefore, adhesins that mediate such adherence are considered to be potential virulence factors [26]. In this study, high prevalence of a number of adhesion-related genes such as ptfA, pfhA, nanB, nanH and hsf-2 were observed except nanH and hsf-1 implying that either these proteins act synergistically or are required at different stages of colonization/infection. Filamentous haemagglutinins encoded by the pfhA gene play a major role in the initial colonization of the upper respiratory tract and the frequency of this gene varies greatly among strains of P. multocida. This gene has been reported to be an important epidemiological marker and correlated with occurrence of disease in cattle, swine and sheep [1,13,27,28]. Interestingly, we observed a very high prevalence of pfhA (95.4%) gene irrespective of its capsule type and origin of isolation. Similar findings (100% prevalence) have also been reported by previous studies on sheep and bovine isolates of India [27,29]. It could be argued that this gene is providing survival advantage to the host and there might have been horizontal gene transfer among isolates resulting in the occurrence of such high prevalence rate among Indian strains. Sialidases (nanB and nanH) not only enhance bacterial virulence by unmasking key host receptors but also reduce the effectiveness of mucin [26 and references therein], thereby playing an active role in colonization of epithelial surface. The prevalence of nanB and nanH varies according to host origins and geographical location [1,9]. In this study, nanB gene was detected in all the isolates, whereas nanH gene was found in 54% of the samples. In contrast, Verma et al. [27], reported 100% prevalence of nanH gene and nonoccurrence of nanB gene among Indian bovine isolates. On the basis of findings from both the studies, it is intriguing to conclude any host specific association between these sialidases genes. Investigation of more isolates from different host origin should be carried out in future to obtain any possible association. Dziva et al. [30] detected toxA gene encoding dermonecrotoxin, only from the samples of serotype D,
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isolated from pigs. Subsequently, it was detected from the strains of serotype A from different hosts [1,13,14,31]. In this study, 48.9% of isolates were found to carry toxA gene. Interestingly, we observed the carriage of this gene in one of each isolate of serotype B and F, which has not been reported to the authors’ knowledge. Probably this is due to the horizontal transfer of gene among isolates as this gene is carried through lysogenic bacteriophage that infects the agent [32]. However, it is puzzling to note if this gene has any role in the pathogenesis or it is a spurious gene. This warrants further investigation. Out of the 88 isolates studied, 53 (60.23%) isolates showed resistance towards at least one antibiotic. Again, out of these 53 isolates, 15 (17.04%) isolates were multidrug resistant (more than 3 drug classes). Most of the isolates of P. multocida were susceptible to enrofloxacin and chloramphenicol followed by tetracycline, ofloxacin, co-trimoxazole, kanamycin, ciprofloxacin, gentamicin and amikacin. Some of the isolates were resistant to ampicillin (38.64%) followed by amoxycillin (22.73%) and erythromycin (15.82%). More or less similar findings have been reported in previous studies except that P. multocida strains were resistant to some of above mentioned antibiotics. This may be due to the preferential administration of some particular antibiotic by the veterinarians during treatment. Almost all previous studies reported that P. multocida recovered from different animals are susceptible to enrofloxacin, chloramphenicol, tetracycline and ciprofloxacin [7,8,28,33]. Kumar et al. [8] found Pasteurella isolates from ruminant origin (cattle, sheep and goat) to have almost similar antibiotic resistance profile with enrofloxacin being most susceptible followed by ofloxacin, chloramphenicol, tetracycline and ciprofloxacin. Recently, Marru et al. [34] reported that ovine isolates causing pneumonic pasteurellosis were highly susceptible to chloramphenicol (100%), sulfamethoxazole (89.1%) and tetracycline (84.4%), while observing complete resistance against gentamicin. 5. Conclusion The present study reports on the carriage of 19 virulence genes and antimicrobial resistance patterns for 17 antibiotics among P. multocida isolates recovered from small ruminants. Our results revealed unique information on frequency of virulence gene i.e., the very high prevalence of pfhA gene. The toxA gene was observed in 48.9% of strains with highest prevalence among serotype A isolates. We also observed this gene in one of each isolate, of capsular type B and F, hitherto unreported. In vitro antibiotic susceptibility test suggested that P. multocida isolates of small ruminant origin were frequently susceptible to enrofloxacin and chloramphenicol. These findings encourage sampling of more isolates in India, representing various hosts to have clearer understanding on the heterogeneity of P. multocida. Acknowledgments Authors are thankful to the Indian Council of Agricultural Research (ICAR), New Delhi, for providing financial support under “All India Network Programme on
Haemorrhagic septicaemia” and the Director, Indian Veterinary Research Institute (IVRI), Izatnagar, for providing facilities to conduct this study. We are thankful to Dr Siba P. Mohanty, Dr Kapileswar Parija of India and Dr Siti Sarah Othmans of Malaysia for critically editing the manuscript. We sincerely thank all the scientists and staff involved in AINP-HS project at IVRI and other collaborating centres who have been instrumental in isolating or maintaining the Pasteurella multocida isolates.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.cimid.2014.11.003.
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Virulence gene profiling and antibiotic resistance pattern of Indian isolates of Pasteurella multocida of small ruminant origin Laxmi N. Sarangi ∗ , P. Thomas, S.K. Gupta, A. Priyadarshini, S. Kumar, Viswas Konasagara Nagaleekar, A. Kumar, Vijendra P. Singh Division of Veterinary Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, UP, India
a r t i c l e
i n f o
Article history: Received 1 January 2014 Received in revised form 11 November 2014 Accepted 20 November 2014 Keywords: Pasteurella multocida Virulence genes Small ruminants toxA pfhA Antibiogram
a b s t r a c t Pasteurellosis in small ruminants affects the livelihood of small and marginal farmers of India. The present study was undertaken to understand the trends in gene carriage and antibiotic resistance pattern of Pasteurella multocida isolates recovered from small ruminants over a period of 10 years in India. A total of 88 P. multocida isolates of small ruminant origin were subjected to virulence gene profiling for 19 genes by PCR and antibiogram study employing 17 different antibiotics. Virulence genes like exbB, exbD, tonB, oma87, sodA, sodC, nanB and plpB (100% prevalence) and ptfA and hsf-2 (>90% prevalence) were found to be uniformly distributed among isolates. Unexpectedly, a very high prevalence (95.45%) of pfhA gene was observed in the present study. Dermonecrotoxin gene (toxA) was observed in 48.9% of isolates with highest occurrence among serotype A isolates and interestingly, one of each isolate of serotype B and F were found to carry this gene. Antimicrobial susceptibility testing revealed 17.04% isolates to be multidrug resistant. Amongst all the antibiotics tested, most of the P. multocida isolates were found to be susceptible to enrofloxacin and chloramphenicol. This study highlights novel epidemiological information on frequency and occurrence of virulence genes among Indian isolates from small ruminants. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Pasteurella multocida belonging to the family Pasteurellaceae is associated with a number of economically important diseases like haemorrhagic septicaemia in cattle and buffalo; enzootic bronchopneumonia in cattle, sheep and goats; atrophic rhinitis in swine; fowl cholera in poultry and snuffles in rabbits [1,2]. In addition, this bacterium has also been isolated from various cases in domestic as well as wild animals [3].
∗ Corresponding author. Tel.: +91 9756597591. E-mail address: [email protected] (L.N. Sarangi). http://dx.doi.org/10.1016/j.cimid.2014.11.003 0147-9571/© 2014 Elsevier Ltd. All rights reserved.
P. multocida has been classified into five types (A, B, D, E and F) based on capsular typing and most often each capsule type has been found to be predominantly associated with a specific disease in a host species [4]. However, over the years, there are reports of occurrence of uncommon capsular type from different host species (reviewed in [2]), which is either due to spontaneous change from one capsular type to another or due to cross species infections [5,6]. Such an ability of the bacteria to colonize, survive and subsequently infect multiple hosts is a great concern, which necessitates a detailed study on isolates from different host origin. In India, animal husbandry practices like cohabitation of various species of animals including poultry and use of common grazing ground may lead to spread of infection among all such host species. This results in a
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complex scenario of circulation of various bacterial strains among members of host populations, notwithstanding an increased possibility of isolates jumping species barriers. Though antibiotics have been used traditionally for the treatment of pasteurellosis, their prolonged and indiscriminate use has led to onset of resistance among various strains [7–9], thus eroding the antibiotic armamentarium and limiting therapeutic options [10]. Most of the antibiotic resistance genes are present on bacterial plasmids, integrons and transposons, which lead to the spread of these genes among isolates [10]. Antimicrobial resistance of Pasteurella isolates varies according to host origin, time of infection, geographical location, anti-bacterial pretreatment and on accessibility of the isolates to the resistance genes present in the gene pool [10]. Hence, it was necessary to carry out a detailed study of the antibiotic susceptibility pattern of P. multocida isolates. Virulence profiling has been used as a typing method for characterization of bacterial pathogens [11] including P. multocida [1,9,12]. However, studies regarding the population structure and virulence gene patterns of isolates from small ruminants are scanty [1,13,14]. Therefore, the present investigation was undertaken to study the population characteristics of P. multocida isolates obtained from small ruminants by determining their virulence-associated gene profiles and phenotypic antibiotic resistance pattern.
2. Materials and methods 2.1. Bacterial isolates Eighty eight (88) P. multocida isolates recovered from small ruminants (sheep, n = 67 and goat, n = 21), maintained at Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar were used in this study. The isolates used in the study were categorized by host species origin, serotyping, year and place of origin. If more than five isolates were obtained from a particular farm/place, then only two representative isolates were selected to be included in the study. The location of the farms (districtwise) and the clinical status of the animal (when available) have been incorporated in the supplementary table.
primers used in the PM-PCR and capsular PCR are given in Table 1. 2.4. Detection of virulence associated genes by PCR All the isolates were screened for carriage of 19 virulence associated genes by uniplex PCR. The PCR was performed with 1 l of DNA, 2.5 l of 10× PCR buffer (containing 20 mM MgCl2 ), 0.5 l of 25 mM MgCl2 , 0.5 l of 10 mM dNTP mix, 1 l each of 10 pM primers, 0.25 l of 5 U Dream Taq DNA polymerase (Fermentas) and the final volume was made up to 25 l using nuclease free water. PCR mixture without DNA was used as negative control. The PCR products were analyzed in 1% agarose gel along with 100 bp DNA ladder (Gene Ruler–Fermentas) by staining with ethidium bromide. The amplified product was visualized under UV light and documented by gel documentation system. The details of the virulence genes and sequences of the oligonucleotide primers are listed in Table 1. 2.5. Antibiotic susceptibility Each of the above isolates were tested for susceptibility against 17 different antibiotics viz. ampicillin (A), amoxycillin (AM), cephalexin (CN), cefotaxime (CE), ceftriaxone (CTR), cephalothin (CH), erythromycin (E), amikacin (AK), kanamycin (K), tetracycline (T), gentamicin (G), ofloxacin (OF), pefloxacin (PF), ciprofloxacin (CF), enrofloxacin (EX), chloramphenicol (C) and co-trimoxazole [sulfamethoxazole with trimethoprim] (CO) using disc diffusion method [19]. These antimicrobials were recommended in OIE terrestrial manual chapter on haemorrhagic septicaemia and/or used by field veterinarians in different parts of India and have proven their clinical efficacy [20–22]. The test was performed by disc diffusion method recommended by Clinical and Laboratory Standards Institute (CLSI; formerly the NCCLS) and the interpretations were carried out as per CLSI standards (Performance Standard for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals M31-A2) [23]. The ranges for susceptible, intermediate and resistant for each drug used in this study are given in Table 2. 2.6. Statistical analysis
2.2. Culture and biochemical tests The isolates were revived by 18–24 h incubation in brain heart infusion (BHI) broth at 37 ◦ C and plated subsequently onto blood agar to study cultural characteristics. They were then tested for purity (biochemical tests: indole, citrate, MR, VP and sugar fermentation tests) as per standard techniques [15].
2.3. Molecular confirmation of P. multocida by P. multocida specific PCR (PM-PCR) and capsular typing The genomic DNA was isolated by CTAB method [16]. The isolates were reconfirmed as P. multocida by PMPCR [17] followed by capsular typing using the primers employed by Townsend et al. [18]. The sequences of the
Statistical analysis of the data generated from the study was performed with SPSS 16.0 (SPSS Inc., Chicago). P values of 90% prevalence). Dermonecrotoxin gene was observed in 48.9% of isolates with highest prevalence among serotype A isolates followed by capsular type D. We observed this gene in one isolate each of serotype B and F (Table 3; Supplementary Table).
In the present study, P. multocida isolates, recovered from small ruminants over a period of years in India, were tested for virulence gene carriage and antibiotic resistance patterns. With the sole exception of capsular type E, all other capsular types have been isolated from small ruminants with majority of the strains from capsular type A followed by B, D and F respectively (Data not shown). Capsular type A strains have been implicated in causing
100 100 100 100 100 93.2 100 50 100 80 54.2 41.7 16.7 83.3 40 64.4 8.3 50 16.7 0 50.8 25.0 50.0 16.7 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 52.5 75.0 50.0 66.7 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 83.1 83.3 100 66.7 40 50.8 58.3 66.7 50.0 60 66.1 91.7 100 83.3 60 94.9 96.6 100 100 100 100 100 100 80 60
69.5 66.7 83.3 66.7 20
89.5 95.2 90.9 49.2 57.1 51.1 53.7 33.3 48.9 49.2 23.8 43.2 100 100 100 100 100 100 100 100 100 100 100 100 56.7 47.6 54.5 100 100 100 100 100 100 100 100 100 83.6 71.4 80.7 49.2 66.7 53.4 73.1 47.6 67.0 73.1 71.4 72.7 95.5 95.2 95.5
NanB NanH Exb D Tonne B Exb B TbpA HasR Positive strains percentage (%)
4. Discussion
No of strains
In this piece of investigation, 35 isolates (39.77%) were found to be susceptible to all the antibiotics under study. Majority of isolates (94%) were susceptible to enrofloxacin and chloramphenicol (Table 4). Species-wise susceptibility pattern suggested that enrofloxacin and chloramphenicol had most of the ovine isolates susceptible to them, followed by tetracycline, gentamicin, ciprofloxacin, ofloxacin and co-trimoxazole. Similarly, most of the caprine isolates were susceptible to enrofloxacin, followed by chloramphenicol, gentamicin, kanamycin, cephalothin, ofloxacin, tetracycline and co-trimoxazole. Among all the antibiotics studied, resistance against ampicillin, amoxycillin and erythromycin were observed in many isolates (Table 4).
Origin
3.3. Antibiogram study
Table 3 Prevalence of virulence associated genes in Pasteurella multocida in respect to host and capsular type.
Chi-square and Fisher’s exact test analysis of association of genes among P. multocida isolates indicated a significant association between pfhA-ptfA, pfhA-hgbB, pfhA-hasR, ptfAhgbB, ptfA-nanH, hgbA-hgbB, hgbA-hasR, hgbA-tbpA, hgbAhsf-1, hgbB-tbpA, hgbB-nanH, hgbB-plpE, tbpA-nanH, tbpAtoxA, plpE-toxA and hsf-1-hsf-2 genes.
SodA
Source: Clinical and Laboratory Standards Institute (CLSI). (Performance Standard for Antimicrobial Disc and Dilution Susceptibility Test for Bacteria Isolated from Animals M31-A2). The interpretation criteria for veterinary pathogens were used. For cephalexin, the interpretation criterion for cefazolin was used as both the antibiotics belonged to same class of antibiotics. Similarly, for cefotaxim and ceftriaxone the criterion for ceftiofur was used and for ofloxacin, pefloxacin and ciprofloxacin the interpretation criterion for enrofloxacin was used. For co-trimoxazole, the interpretation criterion for potentiated sulfonamides (trimethoprimsulfamethoxazole) was used.
95.5 95.2 95.5
Hsf-2 Hsf-1 Tox A
13 13 14 17 17 14 13 14 13 14 12 16 16 16 16 12 10
Plp E
14–16 14–17 15–17 18–20 18–20 15–17 14–22 15–16 14–17 15–18 13–14 17–20 17–20 17–20 17–20 13–17 11–15
Plp B
17 18 18 21 21 18 23 17 18 19 15 21 21 21 21 18 16
Sod C
Ampicillin Amoxycillin Cephalexin Cefotaxime Ceftriaxone Cephalothin Erythromycin Amikacin Kanamycin Tetracyline Gentamicin Ofloxacin Pefloxacin Ciprofloxain Enrofloxacin Chloramphenicol Co-trimoxazole
HgbB
Resistant (≤)
HgbA
Intermediately susceptible
Ptf A
Susceptible (≥)
PfhA
Antibacterial agent
Oma 87
Table 2 Zone diameter interpretation chart (inhibition zone diameter in mm).
100 100 100
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Host origin 67 Sheep 21 Goat Total 88 Capsule type A 59 12 B 6 D 6 F 5 NT
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37
Table 4 Antibiotic susceptibility test result of the Pasteurella multocida isolates in respect to host organism. Host organism
Antibiotic Susceptibility
Sheep (n = 67)
S I R S I R S
Goat (n = 21)
Total (sheep and goat) (n = 88)
I R
Positive strains percentage (%) to individual antibiotics
EX
CN
K
AK
A
AM
CE
E
CTR
CF
PF
T
CO
OF
C
G
CH
94.0 6.0 0 95.2 4.8 0 94.3
70.1 15.0 14.9 81.0 14.3 4.7 72.7
79.1 13.4 7.5 90.5 4.7 4.7 81.8
80.6 14.9 4.5 71.4 23.8 4.8 78.5
34.3 28.7 37.3 42.8 14.3 42.8 36.4
59.7 16.1 23.9 71.4 9.5 19.0 62.5
50.8 35.8 13.4 42.9 52.4 4.7 48.9
28.9 53.7 19.4 23.8 71.4 4.8 26.2
74.6 20.9 4.5 66.7 28.6 4.7 72.7
82.1 13.4 4.5 76.2 19.0 4.8 80.7
74.6 17.9 7.9 66.7 28.6 4.7 77.2
89.5 3.00 7.5 80.9 14.8 4.8 87.5
82.1 8.9 8.9 80.9 4.7 14.8 81.9
82.1 14.9 3.0 85.7 9.5 4.8 82.9
94.0 6.0 0 95.2 4.8 0 94.3
86.6 10.4 3.0 90.5 9.5 0 79.5
77.6 14.9 7.5 85.7 4.8 9.5 79.5
5.7 14.8 0 12.5
11.4 6.8
17.0 4.5
25.0 38.6
14.8 22.7
39.8 11.3
57.9 15.9
22.7 4.5
14.8 4.5
15.9 6.8
5.7 6.8
7.9 10.2
13.6 3.4
5.7 10.2 0 2.3
12.5 8.0
(H = susceptible; I = intermediately susceptible; R = resistant; EX = enrofloxacin; CN = cephalexin; K = kanamycin; AK = amikacin; A = ampicillin; AM = amoxycillin; CE = cefotaxime; E = erythromycin; CTR = ceftriaxone; CF = ciprofloxacin; PF = pefloxacin; T = tetracycline; CO = co-trimoxazole; OF = ofloxacin; C = chloramphenicol; G = gentamicin; CH = cephalothin).
pneumonia in small ruminants [4], but the precise role of other capsular types in disease process is still not clear. Although there are a few reports regarding isolation of serogroup F strains from sheep suffering from respiratory and allied symptoms [24,25], still there is a need for careful analysis of pathogenic potential of these strains [1]. Productive infection by bacterial pathogens relies on the expression of various proteins that have wide ranging functions like competence, adherence, capsule synthesis and evading host immune responses. These proteins or virulence factors are involved in the disease process and are subjected to different selection pressure leading to varying degrees of inter-strain heterogeneity which can be used to assess intra-species diversity and to establish epidemiological relationships [6]. Almost all established virulence associated genes were screened in the present study to obtain comprehensive data on the prevalence of these genes in small ruminant isolates of Indian origin. Genes like ompA, ompH, psl, etc. were not included in this study as previous studies have reported 100% prevalence [1,9]. In P. multocida, different receptors like transferrin receptors (TbpA), heme receptors (HgbA, HgbB, HasR) and Ton B complex (TonB, ExbB, ExbD) are involved in adapting to variations in the supply of different heme iron sources (reviewed in [26]). Among these, TbpA protein has been reported to be an important virulence factor and epidemiological marker in cattle [1,27]. In our study, a high frequency (80.68%) of tbpA was observed, suggesting significant association of this gene with small ruminant isolates. Between two proteins (HgbA and HgbB) involved in acquiring iron directly from the heme component, gene hgbA is present in almost all isolates, whereas prevalence of hgbB varies among strains of different host origin and also with disease status of the animal [1,28]. In contrast to the finding of Shayegh et al. [13] on the low prevalence of hgbB gene among sheep isolates, our study revealed a higher prevalence (67%) of this gene. The frequency of hgbB gene was lower among goat isolates than sheep isolates and this difference between them was found to be statistically significant (p < 0.05). However, due to lack of availability of proper history of majority of the isolates with respect to disease status, it was not possible to perform association
study between virulence gene and disease status of the animal. The basic prerequisite for bacterial infections is its attachment to host cell and therefore, adhesins that mediate such adherence are considered to be potential virulence factors [26]. In this study, high prevalence of a number of adhesion-related genes such as ptfA, pfhA, nanB, nanH and hsf-2 were observed except nanH and hsf-1 implying that either these proteins act synergistically or are required at different stages of colonization/infection. Filamentous haemagglutinins encoded by the pfhA gene play a major role in the initial colonization of the upper respiratory tract and the frequency of this gene varies greatly among strains of P. multocida. This gene has been reported to be an important epidemiological marker and correlated with occurrence of disease in cattle, swine and sheep [1,13,27,28]. Interestingly, we observed a very high prevalence of pfhA (95.4%) gene irrespective of its capsule type and origin of isolation. Similar findings (100% prevalence) have also been reported by previous studies on sheep and bovine isolates of India [27,29]. It could be argued that this gene is providing survival advantage to the host and there might have been horizontal gene transfer among isolates resulting in the occurrence of such high prevalence rate among Indian strains. Sialidases (nanB and nanH) not only enhance bacterial virulence by unmasking key host receptors but also reduce the effectiveness of mucin [26 and references therein], thereby playing an active role in colonization of epithelial surface. The prevalence of nanB and nanH varies according to host origins and geographical location [1,9]. In this study, nanB gene was detected in all the isolates, whereas nanH gene was found in 54% of the samples. In contrast, Verma et al. [27], reported 100% prevalence of nanH gene and nonoccurrence of nanB gene among Indian bovine isolates. On the basis of findings from both the studies, it is intriguing to conclude any host specific association between these sialidases genes. Investigation of more isolates from different host origin should be carried out in future to obtain any possible association. Dziva et al. [30] detected toxA gene encoding dermonecrotoxin, only from the samples of serotype D,
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isolated from pigs. Subsequently, it was detected from the strains of serotype A from different hosts [1,13,14,31]. In this study, 48.9% of isolates were found to carry toxA gene. Interestingly, we observed the carriage of this gene in one of each isolate of serotype B and F, which has not been reported to the authors’ knowledge. Probably this is due to the horizontal transfer of gene among isolates as this gene is carried through lysogenic bacteriophage that infects the agent [32]. However, it is puzzling to note if this gene has any role in the pathogenesis or it is a spurious gene. This warrants further investigation. Out of the 88 isolates studied, 53 (60.23%) isolates showed resistance towards at least one antibiotic. Again, out of these 53 isolates, 15 (17.04%) isolates were multidrug resistant (more than 3 drug classes). Most of the isolates of P. multocida were susceptible to enrofloxacin and chloramphenicol followed by tetracycline, ofloxacin, co-trimoxazole, kanamycin, ciprofloxacin, gentamicin and amikacin. Some of the isolates were resistant to ampicillin (38.64%) followed by amoxycillin (22.73%) and erythromycin (15.82%). More or less similar findings have been reported in previous studies except that P. multocida strains were resistant to some of above mentioned antibiotics. This may be due to the preferential administration of some particular antibiotic by the veterinarians during treatment. Almost all previous studies reported that P. multocida recovered from different animals are susceptible to enrofloxacin, chloramphenicol, tetracycline and ciprofloxacin [7,8,28,33]. Kumar et al. [8] found Pasteurella isolates from ruminant origin (cattle, sheep and goat) to have almost similar antibiotic resistance profile with enrofloxacin being most susceptible followed by ofloxacin, chloramphenicol, tetracycline and ciprofloxacin. Recently, Marru et al. [34] reported that ovine isolates causing pneumonic pasteurellosis were highly susceptible to chloramphenicol (100%), sulfamethoxazole (89.1%) and tetracycline (84.4%), while observing complete resistance against gentamicin. 5. Conclusion The present study reports on the carriage of 19 virulence genes and antimicrobial resistance patterns for 17 antibiotics among P. multocida isolates recovered from small ruminants. Our results revealed unique information on frequency of virulence gene i.e., the very high prevalence of pfhA gene. The toxA gene was observed in 48.9% of strains with highest prevalence among serotype A isolates. We also observed this gene in one of each isolate, of capsular type B and F, hitherto unreported. In vitro antibiotic susceptibility test suggested that P. multocida isolates of small ruminant origin were frequently susceptible to enrofloxacin and chloramphenicol. These findings encourage sampling of more isolates in India, representing various hosts to have clearer understanding on the heterogeneity of P. multocida. Acknowledgments Authors are thankful to the Indian Council of Agricultural Research (ICAR), New Delhi, for providing financial support under “All India Network Programme on
Haemorrhagic septicaemia” and the Director, Indian Veterinary Research Institute (IVRI), Izatnagar, for providing facilities to conduct this study. We are thankful to Dr Siba P. Mohanty, Dr Kapileswar Parija of India and Dr Siti Sarah Othmans of Malaysia for critically editing the manuscript. We sincerely thank all the scientists and staff involved in AINP-HS project at IVRI and other collaborating centres who have been instrumental in isolating or maintaining the Pasteurella multocida isolates.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.cimid.2014.11.003.
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