Curr Microbiol (2014) 68:278–284 DOI 10.1007/s00284-013-0470-y
Pathogenic Providencia alcalifaciens Strain that Causes Fatal Hemorrhagic Pneumonia in Piglets Xinglong Wang • Jinqiu Wang • Huafang Hao Li Qiu • Haijing Liu • Shengli Chen • Ruyi Dang • Zengqi Yang
•
Received: 21 November 2012 / Accepted: 29 August 2013 / Published online: 16 October 2013 Ó Springer Science+Business Media New York 2013
Bacteria from the genus Providencia are opportunistic pathogens, including the six species P. stuartii, P. rettgeri, P. alcalifaciens, P. rustigianii, P. heimbachae, and P. vermicola [16, 24, 30]. Providencia species have been found in humans and animals, and they are also common in
soil, water, and sewage. In humans, Providencia have been isolated from urine (most common), stool, and blood, as well as from the sputum, skin, and wound cultures [5, 24]. In animals, Providencia infections cause neonatal diarrhea in dairy cows (P. stuartii) and enteritis in dogs (P. alcalifaciens) [34]. P. rettgeri has been isolated from crocodiles with meningitis and septicemia [2, 19] and in chickens with enteritis [13]. P. heimbachae has been isolated from penguin feces and an aborted bovine fetus [21]. Although Providencia infections are uncommon and usually nosocomial, the increasing cases of infection have attracted public attention. Recent studies have revealed the link of Providencia spp. with human pericarditis [27], meningitis [28], endocarditis [18], a large outbreak of foodborne illnesses [22], and skin ulceration in dogs [25]. The increasing number of diseases caused by these bacteria indicates their increasing invasiveness. In addition, Providencia spp. isolated from humans, animals, and animal manure has exhibited resistance to various types of antibiotics, including tetracyclines, older penicillins, and cephalosporins [10, 35]. In the current study, we report a strain of P. alcalifaciens that caused fatal hemorrhagic pneumonia in piglets and mice with multi-antibiotic resistance.
Xinglong Wang and Jinqiu Wang equally contributed to this work.
Materials and Methods
X. Wang H. Hao L. Qiu H. Liu S. Chen R. Dang Z. Yang (&) College of Veterinary Medicine, Northwest A&F University, Yangling 712100, People’s Republic of China e-mail: [email protected]
Investigation of Hemorrhagic Pneumonia (HP) in Piglets
Abstract Investigation of a serious pig disease with high mortality and typical lung lesions yielded a bacterial isolate identified as Providencia alcalifaciens based on the 16S ribosomal DNA sequence analysis. The pathogenicity of this bacterial isolate was confirmed in piglets and mice. The bacterial strain caused the typical illness in piglets, which suffered serious dyspnea and hemorrhagic pneumonia. The drug resistance spectrum of the bacterium was also determined. The results indicated that the isolate is resistant to 12 antibiotics and intermediately resistant to 10 antibiotics out of the 34 antibiotics tested. The current study is the first to report a serious lung disease in piglets caused by a multidrug resistant P. alcalifaciens isolate, which should be given more attention during surveillance and diagnostics.
Introduction
J. Wang Department of Animal Husbandry and Veterinary Medicine, Beijing Vocational College of Agriculture, Beijing 102442, China
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A conventional pig-rearing farm in Shaanxi province, China, experienced an outbreak of a respiratory disease. More than 70 % of the suckling piglets died of the disease. Five diseased pigs were euthanized for pathological examination and detection of the pathogens. The gross lesions and necropsy of the piglets were recorded. Two
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samples were collected from each organ, including the heart, liver, spleen, lung, kidney, brain, and lymph node. One sample was homogenized (1 g in 1 ml PBS) for RNA or DNA extraction and bacteria isolation. Another one was preserved in formaldehyde solution for histopathological examination. The RNA and DNA extraction was done following the kit instruction (OMEGA). And 100 ll tissue homogenates were spread on the plates for bacteria isolation. All procedures were approved by the animal care and use committee of Shaanxi province. Etiological Isolation and Inoculum Preparation Several kinds of samples from each piglet were homogenized for detection of PRRSV, PRV, PCV2, and CSFV by PCR or RT-PCR. Meanwhile, the homogenates were used for bacteria isolation. A total of 70 ordinary blood agar plates [nutrient agar (Hope Biol-Technology co., Ltd, Qingdao, China) with 5 % fresh sheep blood] spread with the samples were cultured anaerobically and aerobically at 37 °C for 17–24 h. The grown bacteria were purified three times by picking single colonies, selected based on colony morphology and bacterial staining. The isolates were replicated in nutrient broth medium (Hope Biol-Technology co., Ltd). The cultures in the logarithmic growth phase were washed twice with PBS and stored at -70 °C until use. Amplification and Sequencing of 16S Ribosomal DNA of the Bacterial Isolate Bacterial DNA was extracted following a published protocol [4] and the 16S rDNA was amplified. PCR was performed in a total volume of 25 ll containing 12.5 ll of 29 PCR mix [which contains deoxynucleoside triphosphates (dNTPs), PCR buffer, and Taq polymerase] (Promega), 10 pM primer 16S RNAu (50 -AGAGTTTGATCCT GGCTCAG-30 ), and 10 pM primer 16S RNAd (50 -AAGG AGGTGATCCAGCCGCA-30 ). These primers 16sRNAu and 16sRNAd are universal primers for bacterial 16S rRNA gene targeting of a 1,568 bp amplicon. 2 ll (50 ng) of bacterial template DNA was added to the PCR solution. The sample underwent an initial denaturation step at 95 °C for 5 min, followed by 34 cycles at 95 °C for 1 min, 56 °C for 1 min, 72 °C for 1.5 min, and a final step at 72 °C for 10 min for the last cycle. The PCR products were analyzed by 1 % agarose gel electrophoresis. The clone of the target DNA was done following a previous report [37]. The PCR product was gel purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). The purified DNA was then cloned into pMD-18T vector (Takara, Dalian, China) following the instructions. The positive plasmids identified by restriction enzyme digestion were sent to company for sequencing (Sangon, Shanghai, China).
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Phylogenetic Analysis The phylogenetic data described below were obtained through alignment and phylogenetic analysis of the bacterial 16S rDNA sequences. The nucleotide sequences of the 16S rDNA were aligned, and a neighbor-joining analysis was used to construct phylogenetic trees using MEGA 4 [33]. Experimental Infection in Mice A total of 20 six-week-old male BALB/c mice (purchased from Xi’an Army Hospital University) were equally divided into 4 groups, and then inoculated with the bacterial isolate at 1 9 109 colony-forming units (CFU), 1 9 108 CFU, 1 9 107 CFU, and 1 9 106 CFU in 0.2 ml of sterile phosphate-buffered saline (PBS) per mouse through intraperitoneal (i.p.) injection. The four titers were chosen according to another report researching on the virulence of a clinical source bacteria [3]. Another five mice were assigned as the control and given PBS instead of the bacteria. The livers, spleens, kidneys, and lungs of the mice that killed after experiment of died were collected for bacterial isolation. Experimental Infection in Pigs Six healthy, 15-day-old hybrid piglets (Landrace 9 local stock) were purchased from a local farm. Three of the six piglets were infected with the bacterial isolate through intramuscular (i.m.) injection into their neck muscles, while the remaining three were injected with sterile PBS as the controls. The piglets were housed separately and were fed with milk replacement (Purina, Jiaxing). Each piglet in the infected group received 1 9 1010 CFU of the bacterial isolates in 1 ml of sterile PBS via i.m. injection. The clinical signs of the infected piglets were recorded and necropsies were performed within 1 h after the piglets died. Samples were collected from the lungs, livers, spleens, kidneys, and lymph node (where the bacteria were isolated) of each pig for etiological examination and were plated on ordinary blood agar to isolate bacteria as we did with clinical samples. The colonies on the plate were then identified using API 20E system. Gross lung lesions were observed, and the small lung sheets were fixed in 4 % formalin. Lung sections were made following a previously reported protocol [6], and the histopathologic changes were observed under a microscope. Drug Resistance Test A Kirby–Bauer disk diffusion assay was used to evaluate the resistance of the bacterial isolate. The bacteria were collected with a sterile loop, suspended in peptone water, and incubated at 37 °C for 2 h. The turbidity of the
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suspension was adjusted to 0.5 McFarland’s standard (1.5 9 108 CFU/ml). The suspension was then spread onto the surface of a cation-adjusted Mueller–Hinton agar (MHA) plate using sterile cotton swabs. A total of 34 antibiotic disks with standard antibiotic concentrations were placed on the agar. The plates were then incubated overnight at 37 °C. The zones of inhibition were measured and interpreted based on The Clinical and Laboratory Standards Institute (CLSI) guidelines. The CLSI guidelines were used to classify the isolates as (i) resistant (R), (ii) intermediate resistant (I), and (iii) susceptible (S) to the various antibiotics [38]. Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used as the quality control strains for the susceptibility testing methods.
Results Fatal HPD Occurred in Piglets In the fall of 2011, cases of HPD occurred in the suckling piglets of a conventional pig farm with 500 sows in
Fig. 1 16S rDNA gene sequence dendrogram showing the position of strain shaanxi2011 among recognized Providencia species. Moellerella wisconsensis (AM 040754 and JN175344.1) served as a root.
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Shaanxi province. The infected piglets, which were born healthy, began to show clinical signs as early as 10 days following birth. The clinical symptoms include abdominal skin empurpling, difficulty in breathing, frothy nasal, and oral discharge. The piglets died within 2–4 days after showing clinical signs. The lungs of infected piglets were friable, swollen, and dark brown in color caused by serious hemorrhage. Interstitial broadening and massive inflammatory cells infiltrated can be seen in histologic sections. Etiological Isolation The samples collected from the sick piglets were sent to the laboratory and were used to isolate the pathogen. The PCR results for the detection of PRRSV, PRV, PCV2, and CSFV were negative. However, one bacterial strain (identified by colonial morphology and gram staining) was detected with high proportion on the inoculated plates and which can grow both anaerobically and aerobically. Beside this bacterial strain, several other kinds of bacteria that sporadically appeared in individual piglets were observed, but those were excluded for low grow ratio in plate and were not
Bootstrap values (for 1,000 replicates) are given at each node. The length of the bar represents the variation ratio of the genes
X. Wang et al.: Fatal Hemorrhagic Pneumonia in Piglets
universal. The bacterial strain was considered for etiology and purified for virulence testing on piglets and mice. Polygenic Analysis Based on Partial 16S DNA The 16S rDNA gene sequence of the shaaxi2011 isolate was sequenced and blasted in GenBank. The highest sequence similarity values were among the members of the family Enterobacteriaceae, specifically with genus Providencia ([98 %). The phylogenetically closest type of strain was the Providencia alcalifaciens strain E69 (99.86 % 16S rRNA gene sequence similarity). The sequence of 16S rDNA gene of the shaaxi2011 isolate was submitted to GenBank and the Accession Number is JX827242. Up to 23 16S rRNA gene sequences from Providencia spp., including P. stuartii, P. rettgeri, P. alcalifaciens, P. rustigianii, P. heimbachae, and P. vermicola sp., as well as 2 from Moellerella, were used to construct a phylogenetic tree using the neighbor-joining method using MEGA 4 software (Fig. 1). Although some crosses occurred, the bacterial isolates were classified based on the established polygenic tree. The bacterial isolate shaaxi2011 belonged to P. alcalifaciens and showed high homology with P. alcalifaciens strain E69. Providencia Bacterial Strain Shaaxi2011 was Highly Pathogenic to Piglets and Mice The reproduction of HPD was observed in 15-day-old piglets to determine whether the newly isolated strain of P. alcalifaciens shaaxi2011 was the causative agent of HPD. The piglets inoculated with P. alcalifaciens shaaxi2011 at 1 9 1010 CFU/head through i.m. injection developed serious illness. All piglets inoculated with P. alcalifaciens (n = 3) died within 72 h post-infection. Serious dyspnea was observed before the death of the infected piglets. In addition, the skin on the lower abdomen of the dead piglets were empurpled, which was also a sign of dyspnea. The gross lesions and microscopic lesions in the lungs were examined. The lungs from the infected piglets were swollen and darker in color (Fig. 2). Serious hemorrhage and interstitial broadening were observed based on the histopathologic examination (Fig. 2). The gross lesions and histopathologic lesions observed in the experiment were almost the same as we had seen in clinical samples. The bacteria were recovered from the lungs, lymph nodes, and spleen of the infected piglets as we did with clinical samples. The isolated bacteria showed the same biochemical traits (data not shown); thus, Providencia shaaxi2011 was highly pathogenic to the piglets. To determine whether the pathogenicity of P. alcalifaciens shaaxi2011 against piglets extends to other animals, 6-week-old BALB/c mice were infected with the same
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bacterial isolate through i.p. injection. Fourteen in fifteen of the mice that received doses greater than 1 9 107 CFU/ mouse through i.p. injection died within 24 h, whereas the controls injected with PBS survived (Fig. 3). The same bacteria were reisolated from the samples collected from died mice based on bacteria staining and API 20E biochemical tests results. Therefore, P. alcalifaciens shaaxi2011 is highly pathogenic to piglets and mice. This evidence shows that some strains of Providencia may cause HPD in pigs under experimental conditions. Antimicrobial Resistance Test Of the 34 antibiotics tested, the bacterial isolate was resistant to 12 antibiotics and intermediately resistant to 10 antibiotics (Table 1). Aztreonam and the new generation cephalosporins were effective in inhibiting bacterial growth. Thus, these antibiotics may be useful in controlling the infection.
Discussion Providencia alcalifaciens, a member of the Enterobacteriaceae, has been implicated as an agent of diarrheal disease in children and adults [1, 9, 11]. The role of P. alcalifaciens as an enteric pathogen has been observed previously, and it can invade eukaryotic cell lines [1, 9]. The ability to invade eukaryotic cells was proposed as a virulence mechanism and the invasion ability seems to be strain dependent [9, 14, 29] and even clonally determined [8]. Although P. alcalifaciens is a member of the Enterobacteriaceae, the translocation of this bacteria from the gastrointestinal (GI) tract and its ability to resist human serum bactericidal activity have been reported [36]. Research has also demonstrated the ability of P. alcalifaciens strains to translocate from the intestinal lumen to extraintestinal sites [36]. The current study describes the hemorrhagic pneumonia caused by P. alcalifaciens in piglets. The bacterial isolate was cultured from the lung tissues of each infected piglet. Clinical history, animal testing, and histopathologic examination indicated the role of the bacteria in pathogenesis. The possible mechanism of bacterial invasion in the lungs needs further research. However, the bacteria from Enterobacteriaceae causing extraintestinal infections were quite common; thus, the possible mechanism may be shared. Highland and Sura reported hemorrhagic pneumonia in cats caused by extraintestinal pathogenic E. coli (ExPEC) [12, 32]. ExPEC, distinct from commensal and intestinal pathogenic strains of E. coli, may express cytotoxic necrotizing factors (cnf-1, cnf-2), P-fimbriae adhesins (papG allele I and papG allele III), the major structural subunit of the P-fimbrial shaft
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Fig. 2 Gross lesions and histologic examination of the lungs of the pigs in inoculated (a, c) and control groups (b, d). a The swolling and dark color in the lung were from one of the infected piglets. b The lung was from a piglet of control group. c This is a histologic section
Fig. 3 The survival ratio of BALB/C mice post-infection with P. alcalifaciens shaanxi2011. Twenty-five 6-week-old male BALB/C mice were assigned to five groups with five mice each. Some mice were injected with P. alcalifaciens Shaanxi2011 and some with PBS (as controls), as described in the ‘‘Materials and Methods’’ section. All mice survived in control group, four survived injected with 1 9 106 bacteria, one was kept living in group injected with 1 9 107 bacteria, and all mice died in the other two groups
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from the infected piglet. d This histologic section was from the control group. Hematoxylin and eosin staining (HE). Magnification, 2009
(papA), pilus assembly protein (papC), S fimbriae (sfa), F1C fimbriae (foc), type 1 fimbriae (fim), hemolysin D (hlyD), pathogenicity-associated island marker (malX‘‘PAI’’), novel catecholate siderophore receptor (iroN), Yersinia siderophore receptor (fyuA), genes associated with capsular polysaccharide synthesis (kpsMTII), and outer membrane peptidase (ompT) [15, 17, 26]. The pili or fimbria and fimbrial adhesin molecules promote bacterial adherence and have an important role in colonization in tissue. These factors have been widely researched in E. coli, but not in Providencia spp. Thus, further studies are needed to understand the mechanism of the hemorrhagic pneumonia caused by P. alcalifaciens Shaanxi2011. The members of genus Providencia are commensals in the gastrointestinal tract. However, some species (P. stuartii and P. alcalifaciens) are associated with nosocomial infections in nursing homes and are considered as opportunistic pathogens [23, 34]. Drug resistance in Providencia spp. is of clinical concern because it leads to higher
X. Wang et al.: Fatal Hemorrhagic Pneumonia in Piglets
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Table 1 Drug-resistant spectrum of Providencia alcalifaciens shaanxi2011 Antibiotics
Concentration
Diameter (mm)
Resistance
Antibiotics
Concentration
Diameter (mm)
Resistance
Aztreonam
30 lg
29.1*
S
Ofloxacin
5 lg
15.5
I
Cefotaxime
30 lg
24.9
S
Levofloxacin
5 lg
15.4
I
Cefuroxime
30 lg
23.1
S
Cefoperazone
75 lg
15.2
I
Ceftazidime
30 lg
22.4
S
SMZ-TMP
3.75 lg
14.8
I
Cefepime
30 lg
21.4
S
Chloramphenicol
30 lg
13.9
I
Ceftriaxone
30 lg
21
S
Spectinomycin
100 lg
13
R
Penicillin
10 IU
19.4
I
Oxacillin Sodium
1 lg
11.7
R
Cefoxitin
30 lg
19.3
S
Minocycline
30 lg
11.7
R
Ceftiofur
30 lg
18.6
–
Furantoin
300 lg
12.3
R
Piperacillin
100 lg
18
I
Vancomycin
30 lg
10.1
R
Ampicillin Gentamicin
10 lg 10 lg
18 18
S S
Kanamycin Tetracycline
30 lg 30 lg
9.8 6
R R
Streptomycin
10 lg
18
S
Medemycin
30 lg
6
R
Amikacin
30 lg
16.9
I
Erythrocin
15 lg
6
R
Tobramycin
10 lg
16.7
S
Clarithromycin
15 lg
6
R
Norfloxacin
10 lg
15.8
I
Polymyxin B
300 lg
6
R
Ciprofloxacin
5 lg
15.5
I
Cefazolin
30 lg
6
R
– not given in CLSI, * diameters of inhibition zone
treatment costs and delays in treatment, which could be life threatening. The antibiotic-resistant Providencia spp. strains are important for the spread of resistance by exchanging antibiotic resistance genes with other pathogenic bacterial strains, such as Pseudomonas spp. and Salmonella spp. [7]. Providencia species are generally resistant to tetracyclines, older penicillins, and cephalosporins, and susceptible to late-generation cephalosporins, aztreonam, imipenem, and meropenem. These species also have variable susceptibility to fluoroquinolones, aminoglycosides, and trimethoprim–sulfamethoxazole (TMP– SMX). Each Providencia spp. has different antibiotic resistances. P. stuartii is typically the most resistant Providencia species. Extended spectrum b-lactamase (ESBL)-positive P. stuartii, which comprises 10 % of all ESBL species, has marked resistance to amoxicillin–clavulanate (81.8 %), ampicillin–sulbactam (40.1 %), gentamicin (79.5 %), and ciprofloxacin (84.1 %) based on an Italian survey in 2006 [20]. In another report, 53 % of P. stuartii isolates were found to produce ESBL [35]. P. alcalifaciens and P. rustigianii tend to be the most susceptible Providencia species. Although often resistant to tetracyclines, older penicillins, and cephalosporins, the two species are usually susceptible to TMP-SMX, fluoroquinolones, aminoglycosides, late-generation cephalosporins, aztreonam, and carbapenems [31]. In this study, the P. alcalifaciens isolate was resistant to seven types of antibiotics, including tetracycline antibiotics, macrolides antibiotics, polypeptide antibiotics, cephalosporin antibiotics, penicillinase-resistant b-lactam antibiotics, aminoglycosides
antibiotics, and nitrofuran antibiotics. Based on our drug resistance test, aztreonam was used to treat HPD in the pig farm. After about 1 week’s treatment, about 80 % piglets were cured, about 10 % piglets became stiff pigs, and about 10 % piglets were dead. In summary, an emerging strain of P. alcalifaciens was isolated from diseased piglets with HPD. Fatal HPD was successfully reproduced in piglets using the isolated bacteria, which clearly established that some P. alcalifaciens strains may be the causative agent of HPD in piglets. Thus, P. alcalifaciens is an important zoonotic agent that may have ramifications in human medicine. Acknowledgments This work was funded by the basic research universities special fund operations (QN2011106) and Northwest A&F University PhD Research Start-up funds (2010BSJJ014).
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Pathogenic Providencia alcalifaciens Strain that Causes Fatal Hemorrhagic Pneumonia in Piglets Xinglong Wang • Jinqiu Wang • Huafang Hao Li Qiu • Haijing Liu • Shengli Chen • Ruyi Dang • Zengqi Yang
•
Received: 21 November 2012 / Accepted: 29 August 2013 / Published online: 16 October 2013 Ó Springer Science+Business Media New York 2013
Bacteria from the genus Providencia are opportunistic pathogens, including the six species P. stuartii, P. rettgeri, P. alcalifaciens, P. rustigianii, P. heimbachae, and P. vermicola [16, 24, 30]. Providencia species have been found in humans and animals, and they are also common in
soil, water, and sewage. In humans, Providencia have been isolated from urine (most common), stool, and blood, as well as from the sputum, skin, and wound cultures [5, 24]. In animals, Providencia infections cause neonatal diarrhea in dairy cows (P. stuartii) and enteritis in dogs (P. alcalifaciens) [34]. P. rettgeri has been isolated from crocodiles with meningitis and septicemia [2, 19] and in chickens with enteritis [13]. P. heimbachae has been isolated from penguin feces and an aborted bovine fetus [21]. Although Providencia infections are uncommon and usually nosocomial, the increasing cases of infection have attracted public attention. Recent studies have revealed the link of Providencia spp. with human pericarditis [27], meningitis [28], endocarditis [18], a large outbreak of foodborne illnesses [22], and skin ulceration in dogs [25]. The increasing number of diseases caused by these bacteria indicates their increasing invasiveness. In addition, Providencia spp. isolated from humans, animals, and animal manure has exhibited resistance to various types of antibiotics, including tetracyclines, older penicillins, and cephalosporins [10, 35]. In the current study, we report a strain of P. alcalifaciens that caused fatal hemorrhagic pneumonia in piglets and mice with multi-antibiotic resistance.
Xinglong Wang and Jinqiu Wang equally contributed to this work.
Materials and Methods
X. Wang H. Hao L. Qiu H. Liu S. Chen R. Dang Z. Yang (&) College of Veterinary Medicine, Northwest A&F University, Yangling 712100, People’s Republic of China e-mail: [email protected]
Investigation of Hemorrhagic Pneumonia (HP) in Piglets
Abstract Investigation of a serious pig disease with high mortality and typical lung lesions yielded a bacterial isolate identified as Providencia alcalifaciens based on the 16S ribosomal DNA sequence analysis. The pathogenicity of this bacterial isolate was confirmed in piglets and mice. The bacterial strain caused the typical illness in piglets, which suffered serious dyspnea and hemorrhagic pneumonia. The drug resistance spectrum of the bacterium was also determined. The results indicated that the isolate is resistant to 12 antibiotics and intermediately resistant to 10 antibiotics out of the 34 antibiotics tested. The current study is the first to report a serious lung disease in piglets caused by a multidrug resistant P. alcalifaciens isolate, which should be given more attention during surveillance and diagnostics.
Introduction
J. Wang Department of Animal Husbandry and Veterinary Medicine, Beijing Vocational College of Agriculture, Beijing 102442, China
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A conventional pig-rearing farm in Shaanxi province, China, experienced an outbreak of a respiratory disease. More than 70 % of the suckling piglets died of the disease. Five diseased pigs were euthanized for pathological examination and detection of the pathogens. The gross lesions and necropsy of the piglets were recorded. Two
X. Wang et al.: Fatal Hemorrhagic Pneumonia in Piglets
samples were collected from each organ, including the heart, liver, spleen, lung, kidney, brain, and lymph node. One sample was homogenized (1 g in 1 ml PBS) for RNA or DNA extraction and bacteria isolation. Another one was preserved in formaldehyde solution for histopathological examination. The RNA and DNA extraction was done following the kit instruction (OMEGA). And 100 ll tissue homogenates were spread on the plates for bacteria isolation. All procedures were approved by the animal care and use committee of Shaanxi province. Etiological Isolation and Inoculum Preparation Several kinds of samples from each piglet were homogenized for detection of PRRSV, PRV, PCV2, and CSFV by PCR or RT-PCR. Meanwhile, the homogenates were used for bacteria isolation. A total of 70 ordinary blood agar plates [nutrient agar (Hope Biol-Technology co., Ltd, Qingdao, China) with 5 % fresh sheep blood] spread with the samples were cultured anaerobically and aerobically at 37 °C for 17–24 h. The grown bacteria were purified three times by picking single colonies, selected based on colony morphology and bacterial staining. The isolates were replicated in nutrient broth medium (Hope Biol-Technology co., Ltd). The cultures in the logarithmic growth phase were washed twice with PBS and stored at -70 °C until use. Amplification and Sequencing of 16S Ribosomal DNA of the Bacterial Isolate Bacterial DNA was extracted following a published protocol [4] and the 16S rDNA was amplified. PCR was performed in a total volume of 25 ll containing 12.5 ll of 29 PCR mix [which contains deoxynucleoside triphosphates (dNTPs), PCR buffer, and Taq polymerase] (Promega), 10 pM primer 16S RNAu (50 -AGAGTTTGATCCT GGCTCAG-30 ), and 10 pM primer 16S RNAd (50 -AAGG AGGTGATCCAGCCGCA-30 ). These primers 16sRNAu and 16sRNAd are universal primers for bacterial 16S rRNA gene targeting of a 1,568 bp amplicon. 2 ll (50 ng) of bacterial template DNA was added to the PCR solution. The sample underwent an initial denaturation step at 95 °C for 5 min, followed by 34 cycles at 95 °C for 1 min, 56 °C for 1 min, 72 °C for 1.5 min, and a final step at 72 °C for 10 min for the last cycle. The PCR products were analyzed by 1 % agarose gel electrophoresis. The clone of the target DNA was done following a previous report [37]. The PCR product was gel purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). The purified DNA was then cloned into pMD-18T vector (Takara, Dalian, China) following the instructions. The positive plasmids identified by restriction enzyme digestion were sent to company for sequencing (Sangon, Shanghai, China).
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Phylogenetic Analysis The phylogenetic data described below were obtained through alignment and phylogenetic analysis of the bacterial 16S rDNA sequences. The nucleotide sequences of the 16S rDNA were aligned, and a neighbor-joining analysis was used to construct phylogenetic trees using MEGA 4 [33]. Experimental Infection in Mice A total of 20 six-week-old male BALB/c mice (purchased from Xi’an Army Hospital University) were equally divided into 4 groups, and then inoculated with the bacterial isolate at 1 9 109 colony-forming units (CFU), 1 9 108 CFU, 1 9 107 CFU, and 1 9 106 CFU in 0.2 ml of sterile phosphate-buffered saline (PBS) per mouse through intraperitoneal (i.p.) injection. The four titers were chosen according to another report researching on the virulence of a clinical source bacteria [3]. Another five mice were assigned as the control and given PBS instead of the bacteria. The livers, spleens, kidneys, and lungs of the mice that killed after experiment of died were collected for bacterial isolation. Experimental Infection in Pigs Six healthy, 15-day-old hybrid piglets (Landrace 9 local stock) were purchased from a local farm. Three of the six piglets were infected with the bacterial isolate through intramuscular (i.m.) injection into their neck muscles, while the remaining three were injected with sterile PBS as the controls. The piglets were housed separately and were fed with milk replacement (Purina, Jiaxing). Each piglet in the infected group received 1 9 1010 CFU of the bacterial isolates in 1 ml of sterile PBS via i.m. injection. The clinical signs of the infected piglets were recorded and necropsies were performed within 1 h after the piglets died. Samples were collected from the lungs, livers, spleens, kidneys, and lymph node (where the bacteria were isolated) of each pig for etiological examination and were plated on ordinary blood agar to isolate bacteria as we did with clinical samples. The colonies on the plate were then identified using API 20E system. Gross lung lesions were observed, and the small lung sheets were fixed in 4 % formalin. Lung sections were made following a previously reported protocol [6], and the histopathologic changes were observed under a microscope. Drug Resistance Test A Kirby–Bauer disk diffusion assay was used to evaluate the resistance of the bacterial isolate. The bacteria were collected with a sterile loop, suspended in peptone water, and incubated at 37 °C for 2 h. The turbidity of the
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suspension was adjusted to 0.5 McFarland’s standard (1.5 9 108 CFU/ml). The suspension was then spread onto the surface of a cation-adjusted Mueller–Hinton agar (MHA) plate using sterile cotton swabs. A total of 34 antibiotic disks with standard antibiotic concentrations were placed on the agar. The plates were then incubated overnight at 37 °C. The zones of inhibition were measured and interpreted based on The Clinical and Laboratory Standards Institute (CLSI) guidelines. The CLSI guidelines were used to classify the isolates as (i) resistant (R), (ii) intermediate resistant (I), and (iii) susceptible (S) to the various antibiotics [38]. Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used as the quality control strains for the susceptibility testing methods.
Results Fatal HPD Occurred in Piglets In the fall of 2011, cases of HPD occurred in the suckling piglets of a conventional pig farm with 500 sows in
Fig. 1 16S rDNA gene sequence dendrogram showing the position of strain shaanxi2011 among recognized Providencia species. Moellerella wisconsensis (AM 040754 and JN175344.1) served as a root.
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Shaanxi province. The infected piglets, which were born healthy, began to show clinical signs as early as 10 days following birth. The clinical symptoms include abdominal skin empurpling, difficulty in breathing, frothy nasal, and oral discharge. The piglets died within 2–4 days after showing clinical signs. The lungs of infected piglets were friable, swollen, and dark brown in color caused by serious hemorrhage. Interstitial broadening and massive inflammatory cells infiltrated can be seen in histologic sections. Etiological Isolation The samples collected from the sick piglets were sent to the laboratory and were used to isolate the pathogen. The PCR results for the detection of PRRSV, PRV, PCV2, and CSFV were negative. However, one bacterial strain (identified by colonial morphology and gram staining) was detected with high proportion on the inoculated plates and which can grow both anaerobically and aerobically. Beside this bacterial strain, several other kinds of bacteria that sporadically appeared in individual piglets were observed, but those were excluded for low grow ratio in plate and were not
Bootstrap values (for 1,000 replicates) are given at each node. The length of the bar represents the variation ratio of the genes
X. Wang et al.: Fatal Hemorrhagic Pneumonia in Piglets
universal. The bacterial strain was considered for etiology and purified for virulence testing on piglets and mice. Polygenic Analysis Based on Partial 16S DNA The 16S rDNA gene sequence of the shaaxi2011 isolate was sequenced and blasted in GenBank. The highest sequence similarity values were among the members of the family Enterobacteriaceae, specifically with genus Providencia ([98 %). The phylogenetically closest type of strain was the Providencia alcalifaciens strain E69 (99.86 % 16S rRNA gene sequence similarity). The sequence of 16S rDNA gene of the shaaxi2011 isolate was submitted to GenBank and the Accession Number is JX827242. Up to 23 16S rRNA gene sequences from Providencia spp., including P. stuartii, P. rettgeri, P. alcalifaciens, P. rustigianii, P. heimbachae, and P. vermicola sp., as well as 2 from Moellerella, were used to construct a phylogenetic tree using the neighbor-joining method using MEGA 4 software (Fig. 1). Although some crosses occurred, the bacterial isolates were classified based on the established polygenic tree. The bacterial isolate shaaxi2011 belonged to P. alcalifaciens and showed high homology with P. alcalifaciens strain E69. Providencia Bacterial Strain Shaaxi2011 was Highly Pathogenic to Piglets and Mice The reproduction of HPD was observed in 15-day-old piglets to determine whether the newly isolated strain of P. alcalifaciens shaaxi2011 was the causative agent of HPD. The piglets inoculated with P. alcalifaciens shaaxi2011 at 1 9 1010 CFU/head through i.m. injection developed serious illness. All piglets inoculated with P. alcalifaciens (n = 3) died within 72 h post-infection. Serious dyspnea was observed before the death of the infected piglets. In addition, the skin on the lower abdomen of the dead piglets were empurpled, which was also a sign of dyspnea. The gross lesions and microscopic lesions in the lungs were examined. The lungs from the infected piglets were swollen and darker in color (Fig. 2). Serious hemorrhage and interstitial broadening were observed based on the histopathologic examination (Fig. 2). The gross lesions and histopathologic lesions observed in the experiment were almost the same as we had seen in clinical samples. The bacteria were recovered from the lungs, lymph nodes, and spleen of the infected piglets as we did with clinical samples. The isolated bacteria showed the same biochemical traits (data not shown); thus, Providencia shaaxi2011 was highly pathogenic to the piglets. To determine whether the pathogenicity of P. alcalifaciens shaaxi2011 against piglets extends to other animals, 6-week-old BALB/c mice were infected with the same
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bacterial isolate through i.p. injection. Fourteen in fifteen of the mice that received doses greater than 1 9 107 CFU/ mouse through i.p. injection died within 24 h, whereas the controls injected with PBS survived (Fig. 3). The same bacteria were reisolated from the samples collected from died mice based on bacteria staining and API 20E biochemical tests results. Therefore, P. alcalifaciens shaaxi2011 is highly pathogenic to piglets and mice. This evidence shows that some strains of Providencia may cause HPD in pigs under experimental conditions. Antimicrobial Resistance Test Of the 34 antibiotics tested, the bacterial isolate was resistant to 12 antibiotics and intermediately resistant to 10 antibiotics (Table 1). Aztreonam and the new generation cephalosporins were effective in inhibiting bacterial growth. Thus, these antibiotics may be useful in controlling the infection.
Discussion Providencia alcalifaciens, a member of the Enterobacteriaceae, has been implicated as an agent of diarrheal disease in children and adults [1, 9, 11]. The role of P. alcalifaciens as an enteric pathogen has been observed previously, and it can invade eukaryotic cell lines [1, 9]. The ability to invade eukaryotic cells was proposed as a virulence mechanism and the invasion ability seems to be strain dependent [9, 14, 29] and even clonally determined [8]. Although P. alcalifaciens is a member of the Enterobacteriaceae, the translocation of this bacteria from the gastrointestinal (GI) tract and its ability to resist human serum bactericidal activity have been reported [36]. Research has also demonstrated the ability of P. alcalifaciens strains to translocate from the intestinal lumen to extraintestinal sites [36]. The current study describes the hemorrhagic pneumonia caused by P. alcalifaciens in piglets. The bacterial isolate was cultured from the lung tissues of each infected piglet. Clinical history, animal testing, and histopathologic examination indicated the role of the bacteria in pathogenesis. The possible mechanism of bacterial invasion in the lungs needs further research. However, the bacteria from Enterobacteriaceae causing extraintestinal infections were quite common; thus, the possible mechanism may be shared. Highland and Sura reported hemorrhagic pneumonia in cats caused by extraintestinal pathogenic E. coli (ExPEC) [12, 32]. ExPEC, distinct from commensal and intestinal pathogenic strains of E. coli, may express cytotoxic necrotizing factors (cnf-1, cnf-2), P-fimbriae adhesins (papG allele I and papG allele III), the major structural subunit of the P-fimbrial shaft
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Fig. 2 Gross lesions and histologic examination of the lungs of the pigs in inoculated (a, c) and control groups (b, d). a The swolling and dark color in the lung were from one of the infected piglets. b The lung was from a piglet of control group. c This is a histologic section
Fig. 3 The survival ratio of BALB/C mice post-infection with P. alcalifaciens shaanxi2011. Twenty-five 6-week-old male BALB/C mice were assigned to five groups with five mice each. Some mice were injected with P. alcalifaciens Shaanxi2011 and some with PBS (as controls), as described in the ‘‘Materials and Methods’’ section. All mice survived in control group, four survived injected with 1 9 106 bacteria, one was kept living in group injected with 1 9 107 bacteria, and all mice died in the other two groups
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from the infected piglet. d This histologic section was from the control group. Hematoxylin and eosin staining (HE). Magnification, 2009
(papA), pilus assembly protein (papC), S fimbriae (sfa), F1C fimbriae (foc), type 1 fimbriae (fim), hemolysin D (hlyD), pathogenicity-associated island marker (malX‘‘PAI’’), novel catecholate siderophore receptor (iroN), Yersinia siderophore receptor (fyuA), genes associated with capsular polysaccharide synthesis (kpsMTII), and outer membrane peptidase (ompT) [15, 17, 26]. The pili or fimbria and fimbrial adhesin molecules promote bacterial adherence and have an important role in colonization in tissue. These factors have been widely researched in E. coli, but not in Providencia spp. Thus, further studies are needed to understand the mechanism of the hemorrhagic pneumonia caused by P. alcalifaciens Shaanxi2011. The members of genus Providencia are commensals in the gastrointestinal tract. However, some species (P. stuartii and P. alcalifaciens) are associated with nosocomial infections in nursing homes and are considered as opportunistic pathogens [23, 34]. Drug resistance in Providencia spp. is of clinical concern because it leads to higher
X. Wang et al.: Fatal Hemorrhagic Pneumonia in Piglets
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Table 1 Drug-resistant spectrum of Providencia alcalifaciens shaanxi2011 Antibiotics
Concentration
Diameter (mm)
Resistance
Antibiotics
Concentration
Diameter (mm)
Resistance
Aztreonam
30 lg
29.1*
S
Ofloxacin
5 lg
15.5
I
Cefotaxime
30 lg
24.9
S
Levofloxacin
5 lg
15.4
I
Cefuroxime
30 lg
23.1
S
Cefoperazone
75 lg
15.2
I
Ceftazidime
30 lg
22.4
S
SMZ-TMP
3.75 lg
14.8
I
Cefepime
30 lg
21.4
S
Chloramphenicol
30 lg
13.9
I
Ceftriaxone
30 lg
21
S
Spectinomycin
100 lg
13
R
Penicillin
10 IU
19.4
I
Oxacillin Sodium
1 lg
11.7
R
Cefoxitin
30 lg
19.3
S
Minocycline
30 lg
11.7
R
Ceftiofur
30 lg
18.6
–
Furantoin
300 lg
12.3
R
Piperacillin
100 lg
18
I
Vancomycin
30 lg
10.1
R
Ampicillin Gentamicin
10 lg 10 lg
18 18
S S
Kanamycin Tetracycline
30 lg 30 lg
9.8 6
R R
Streptomycin
10 lg
18
S
Medemycin
30 lg
6
R
Amikacin
30 lg
16.9
I
Erythrocin
15 lg
6
R
Tobramycin
10 lg
16.7
S
Clarithromycin
15 lg
6
R
Norfloxacin
10 lg
15.8
I
Polymyxin B
300 lg
6
R
Ciprofloxacin
5 lg
15.5
I
Cefazolin
30 lg
6
R
– not given in CLSI, * diameters of inhibition zone
treatment costs and delays in treatment, which could be life threatening. The antibiotic-resistant Providencia spp. strains are important for the spread of resistance by exchanging antibiotic resistance genes with other pathogenic bacterial strains, such as Pseudomonas spp. and Salmonella spp. [7]. Providencia species are generally resistant to tetracyclines, older penicillins, and cephalosporins, and susceptible to late-generation cephalosporins, aztreonam, imipenem, and meropenem. These species also have variable susceptibility to fluoroquinolones, aminoglycosides, and trimethoprim–sulfamethoxazole (TMP– SMX). Each Providencia spp. has different antibiotic resistances. P. stuartii is typically the most resistant Providencia species. Extended spectrum b-lactamase (ESBL)-positive P. stuartii, which comprises 10 % of all ESBL species, has marked resistance to amoxicillin–clavulanate (81.8 %), ampicillin–sulbactam (40.1 %), gentamicin (79.5 %), and ciprofloxacin (84.1 %) based on an Italian survey in 2006 [20]. In another report, 53 % of P. stuartii isolates were found to produce ESBL [35]. P. alcalifaciens and P. rustigianii tend to be the most susceptible Providencia species. Although often resistant to tetracyclines, older penicillins, and cephalosporins, the two species are usually susceptible to TMP-SMX, fluoroquinolones, aminoglycosides, late-generation cephalosporins, aztreonam, and carbapenems [31]. In this study, the P. alcalifaciens isolate was resistant to seven types of antibiotics, including tetracycline antibiotics, macrolides antibiotics, polypeptide antibiotics, cephalosporin antibiotics, penicillinase-resistant b-lactam antibiotics, aminoglycosides
antibiotics, and nitrofuran antibiotics. Based on our drug resistance test, aztreonam was used to treat HPD in the pig farm. After about 1 week’s treatment, about 80 % piglets were cured, about 10 % piglets became stiff pigs, and about 10 % piglets were dead. In summary, an emerging strain of P. alcalifaciens was isolated from diseased piglets with HPD. Fatal HPD was successfully reproduced in piglets using the isolated bacteria, which clearly established that some P. alcalifaciens strains may be the causative agent of HPD in piglets. Thus, P. alcalifaciens is an important zoonotic agent that may have ramifications in human medicine. Acknowledgments This work was funded by the basic research universities special fund operations (QN2011106) and Northwest A&F University PhD Research Start-up funds (2010BSJJ014).
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