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Article

Antimicrobial Resistance (AMR) of Bacteria Isolated from Dogs with Canine Parvovirus (CPV) Infection: The Need for a Rational Use of Antibiotics in Companion Animal Health

by
Giorgia Schirò
†,
Delia Gambino
†,
Francesco Mira
*,
Maria Vitale
,
Annalisa Guercio
,
Giuseppa Purpari
,
Francesco Antoci
,
Francesca Licitra
,
Gabriele Chiaramonte
,
Maria La Giglia
,
Vincenzo Randazzo
and
Domenico Vicari
Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Via Gino Marinuzzi n. 3, 90129 Palermo, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Antibiotics 2022, 11(2), 142; https://doi.org/10.3390/antibiotics11020142
Submission received: 23 November 2021 / Revised: 17 January 2022 / Accepted: 19 January 2022 / Published: 23 January 2022
(This article belongs to the Special Issue Monitoring and Surveillance of Veterinary Antimicrobial Use and Antibiotic Resistance in Animals)
Versions Notes

Abstract

:
Canine parvovirus type 2 (CPV-2) represents a major viral threat to dogs. Considering the potential effects of pets on antimicrobial resistance, information on the CPV and associated bacterial co-infections is limited. The aim of this study was to analyze the antimicrobial susceptibility and multidrug-resistance profiles of bacterial species from tissue samples of dogs with canine parvovirus infection. A set of PCR assays and sequence analyses was used for the detection and the molecular characterization of the CPV strains and other enteric viruses. Bacterial isolation, the determination of antimicrobial susceptibility via the disk diffusion method, and the determination of the minimum inhibitory concentration were performed. The detection of β-lactamase genes and toxin genes for specific bacteria was also carried out. CPV infection was confirmed in 23 dogs. Forty-three bacterial strains were isolated and all showed phenotypic resistance. Seventeen multidrug-resistant bacteria and bacteria with high resistance to third- and fourth-generation cephalosporins and metronidazole were detected. Almost 50% of the isolated Enterobacteriaceae were positive for at least one β-lactamase gene, with the majority carrying more genes as well. The evidence for multi-resistant bacteria with the potential for intra- or cross-species transmission should be further considered in a One Health approach.

1. Introduction

Canine parvovirus type 2 (CPV-2) emerged as a dog pathogen in early 1978 and rapidly spread worldwide, causing a pandemic event [1]. Despite the widespread use of effective vaccines, after forty years, CPV remains a significant pathogen, still representing a major threat to young dogs [2,3,4].
Canine parvovirus (CPV) is a small (about 25 nm in diameter), non-enveloped DNA virus, recently included with other related parvoviruses in the unique species Carnivore protoparvovirus 1, within the Protoparvovirus genus (family Parvoviridae, subfamily Parvovirinae) [5,6,7]. Soon after its emergence, two antigenic variants (CPV-2a and CPV-2b) were identified, replacing the original CPV-2 type [8,9]. In 2000, a third antigenic variant (CPV-2c) rapidly spread, and all three variants are currently distributed worldwide with different prevalence rates [5,10].
Parvoviral infection is characterized by depression, anorexia, vomiting, and severe enteritis; mucoid or bloody diarrhea, dehydration, leukopenia, fever, and shock are also detected [5,11]. No specific therapy exists for CPV, and therefore, treatment is primarily supportive and symptomatic, mainly based on rehydration and antimicrobial and antiemetic therapies [11,12,13].
Alterations of the intestinal mucosa have been associated with CPV infection [14], leading to the disruption of gut barrier function and microbiota dysbiosis [15,16,17]. These changes can result in bacterial and endotoxin translocation, with the consequent development of systemic inflammatory responses and multiple organ dysfunction syndromes [11]. A broad spectrum of antibiotics is used in the therapy of CPV infection, although the use of antibiotics may cause an increase in the release of endotoxins and/or the exacerbation of the systemic inflammatory response [14,18]. Despite the close relationship between CPV infection and the bacterial population, few studies on these co-infections are currently available [19,20,21,22].
Moreover, the diffuse and sometimes uncontrolled use of antibiotics in veterinary medicine has increased concerns related to the high diffusion of antimicrobial resistance (AMR), a real threat to public health in all countries. For the efficacious control of AMR spread worldwide, a One Health perspective has been suggested by the international authorities for public health [23]. Particularly, attention is focused on those critically important antimicrobials (CIAs) for human medicine [24]. Indeed, there is considerable evidence that their use in animals can also contribute to antimicrobial resistance among some common enteric human pathogens [25,26]. Most of this renewed attention is focused on food-producing animals, as well as their potential role in environmental contamination with AMR strains; however, a small—but increasing—amount of the current research is also looking at the potential role of pets.
The aim of this study was the evaluation of the antimicrobial susceptibility and multidrug resistance (MDR) profiles of bacterial species from the tissue samples of 23 dogs with canine parvovirus infection. Co-infection with other viruses was also analyzed.

2. Results

2.1. Clinical Cases

All 23 sampled dog carcasses, with a modal age value of 2 months (ranging from 40 days to 2 years), were submitted for suspected infectious gastrointestinal disease. Most of them (n = 16) were young dogs (<12 months) and mixed breed stray (n = 15) dogs (Supplementary Materials Table S1). Anamnesis, the clinical history, and the vaccination statuses of most of the analyzed dogs were unavailable.
At necropsy, the common anatomopathological lesions characteristic of CPV infection were observed: hyperemia of the gastric mucosa and catarrhal-hemorrhagic fluids in the stomach, hyperemia, and hemorrhage of the serous membrane of the small intestine, and congestion and enlargement of mesenteric lymph nodes. In some dogs, further lesions (brain edema and hemorrhage, paleness of and focal fibrous lesions on the myocardium, ecchymoses, petechiae and necrosis on the lungs, subcutaneous petechiae, and icterus and hepatic lipidosis) in different organs were observed (Supplementary Materials Figure S1).

2.2. Viral Detection and Molecular Characterization of CPV

All the tissue samples tested positive for CPV by conventional PCR assay. Positive samples were obtained both from commonly and from less tested tissues, such as the brain and cerebellum, bone marrow [27], and spinal cord. The tissue samples tested negative for canine distemper virus (CDV), canine adenovirus (CAdV) type 2, and canine rotavirus (CRoV), with the exception of five intestines, which tested positive for canine coronavirus (CCoV), and the tissues of dog id. 19, which tested positive for CAdV type 1 (Supplementary Materials Table S1).
Based on the analysis of the VP2 amino acid residues, 9, 3, and 11 CPV strains were typed as CPV2a, CPV-2b, and CPV-2c variants, respectively.
The phylogenetic analysis (Supplementary Materials Figure S2) evidenced the relationship with CPV-2a/2b/2c strains previously reported in Italy [28,29], as well as with CPV-2c strains more recently circulating in Italy and in Asia [30,31]. The viral variants are listed in Supplementary Materials Table S1.

2.3. Bacterial Detection

The bacteriological examination was carried out on 161 tissue samples. One or more bacterial species were isolated from all the dogs, for a total of 43 strains, mainly from the intestine but also frequently from the brain, liver, spleen, heart, kidney, and, less frequently, lung and lymph nodes (Table 1, Supplementary Materials Table S1). The most isolated bacterial species (n = 31) belong to the Gram-negative group (72%), with the highest prevalence at the species level represented by Escherichia coli (19/43, 44%). Klebsiella pneumoniae (4/43, 9.3%), Enterobacter spp. (4/43, 9.3%), Escherichia fergusonii (1/43, 2.3%), Salmonella enterica subsp. Enterica serovar Schleissheim (1/43, 2.3%), Proteus mirabilis (1/43, 2.3%), and Pseudomonas aeruginosa (1/43, 2.3%) were also detected. Among the Gram-positive bacteria (n = 12), equal amounts of strains belonging to the genuses Enterococcus (3/43, 6.9%), Staphylococcus (3/43, 6.9%), Streptococcus (3/43, 6.9%), and Clostridium (3/43, 6.9%) were isolated.
A unique bacterial species was isolated in 43% (10/23) of the dogs, whereas in 22% (5/23) and 26% (6/23), two and three bacterial species were simultaneously isolated, respectively. The coexistence of four different bacterial species was detected only in one dog (id. 20). Although they are normally present in the intestinal microbiota, E. coli strains were isolated from the intestines of only 7/23 dogs. In 12/23, E. coli was also isolated from other organs. The isolated bacterial species are listed in Table 1 and Supplementary Materials Table S1.

2.4. Antimicrobial Susceptibility According to the Disk Diffusion Method

The results obtained with the Kirby-Bauer method showed the presence of resistance in all 43 isolated strains. All the Gram-negative strains (31/43, 72%) were resistant to cefquinome (fourth gen. cephalosporin), methicillin, and metronidazole. Some strains were resistant to antibiotics considered the last line of defense against resistant infections in human health: Escherichia fergusonii was resistant to colistin sulphate, one E. coli to imipenem, and three E. coli were resistant to chloramphenicol. Two more E. coli with two Klebsiella pneumoniae strains and one Enterobacter cloacae strain were resistant to ceftriaxone. Two E. coli isolated from dogs 19 and 22 were simultaneously resistant to chloramphenicol/ceftriaxone and chloramphenicol/imipenem, respectively. Additionally, another five bacteria (four E. coli and a Pseudomonas aeruginosa) showed intermediate sensitivity to imipenem, and one more E. coli isolate to chloramphenicol. In addition, many of the strains of Enterobacteriaceae were resistant to cefadroxil and cephalexin (first gen. cephalosporin). All the Klebsiella pneumoniae (4/31), one of two Enterobacter gergoviae (2/31), and Salmonella enterica (1/31) were found to be resistant to cefadroxil and cephalexin, and one Enterobacter gergoviae (2/31) was resistant to cefadroxil and sensitive to cefalexin, while of the 19 E. coli isolated, 15 were resistant to cephalexin and 14 to cefadroxil.
On the other hand, most of the Gram-negative strains were sensitive to marbofloxacin (24/31, 77.4%), enrofloxacin, and sulfamethoxazole/trimethoprim (19/31, 61.2%). Despite the reported intrinsic resistance [32], one Enterobacter cloacae and the Pseudomonas aeruginosa strains tested sensitive to penicillin, chloramphenicol, and doxycycline. The antibiotic sensitivity results for the Gram-negative strains are shown in Table 2.
Most of the Gram-positive strains were resistant to metronidazole (10/12, 83.3%), cefquinome (fourth gen. cephalosporin) (9/12, 75%), and cefuroxime (second gen. cephalosporin) (6/12, 50%) and sensitive to vancomycin (12/12, 100%), amoxicillin/clavulanic acid, and imipenem (10/12, 83.3%), marbofloxacin (8/12, 66.6%), cephalexin, cefadroxil, and ceftriaxone (first and third gen. cephalosporin, respectively) (7/12, 58.3%). The antibiotic sensitivity results for the Gram-positive strains are shown in Table 3.

2.5. Antimicrobial Susceptibility According to the Minimum Inhibitory Concentration (MIC)

To quantitatively assess the bacterial sensitivity to some of the antibiotics previously tested with the Kirby–Bauer test, five sets of antibiotics were used to determine the MICs; additional molecules were selected by the manufacturer according to the bacterial species. The MICs are reported in Supplementary Materials Tables S2 and S3.
The set used for the 28 Gram-negative bacteria confirmed the higher incidence of resistance to cephalexin (77.7% of the 27 strains tested) and ampicillin (47.3% of the 19 strains tested): 15/28 E. coli, 2/28 Enterobacter cloacae, 2/28 Enterobacter gergoviae, and 2 Klebsiella pneumoniae were resistant to cephalexin (first gen. cephalosporin) and 9/28 E. coli to ampicillin. Some antibiotics were tested only by the MIC assay, and the following results were derived: 8/28 E. coli, 4/28 Klebsiella pneumoniae, and 1/28 Enterobacter cloacae were found to be resistant to piperacillin (46.4%); 6/28 E. coli, 4/28 Klebsiella pneumoniae, 2/28 Enterobacter cloacae, and 1/28 Proteus mirabilis to tetracycline (48.1% of the 27 strains tested). All of them were sensitive to amikacin, and many, to marbofloxacin (24/28, 85.7%), tobramycin (22/28, 78.5%), enrofloxacin, and gentamicin (21/28, 75%) as well. The two isolates, E. fergusonii and S. enterica, showed sensitivity to all the tested antibiotics.
Among the Gram-positive strains, the MICs determined for 1/8 Streptococcus pseudoporcinus showed sensitivity to all the tested antibiotics, whereas 1/8 Streptococcus canis was resistant to tetracycline only. However, resistance was found in the strains of Enterococcus faecium and Staphylococcus xylosus: both Enterococcus faecium strains tested were resistant to enrofloxacin, marbofloxacin, and doxycycline, and one of them, also to erythromycin, while the Staphylococcus xylosus strain tested was resistant to clindamycin, enrofloxacin, marbofloxacin, doxycycline, and minocycline. Moreover, all the Gram-positive strains have been found to be sensitive to chloramphenicol and florphenicol.

2.6. Molecular Analysis of β-Lactamase Genes

All the Enterobacteriaceae (30 isolates in Table 4) from the dogs were further analyzed for the presence of β-lactamase genes. The results show that almost 50% (15/32) of the isolates were positive, according to PCR, for at least one β-lactamase gene, with the majority also carrying more genes simultaneously. Of the 15 β-lactamase-positive strains, 11 isolates carried the blaTEM gene and nine the blaCTXM-II gene.
Some isolates of E. coli tested positive for at least one β-lactamase gene (8/19). All but one carried the blaTEM gene, mostly associated with other genes. Only one carried just blaOXA and blaCTX-M-II, and one, only blaTEM (Table 4). Klebsiella pneumonia was also isolated from four mixed breed dogs (three strays and one owned). Of these isolates, two carried four β-lactamase genes simultaneously (blaSHV, blaOXA, blaTEM, and blaCTXM-II), one carried three genes (blaSHV, blaTEM, and blaCTXM-II), and one carried only the blaSHV gene, normally present in all K. pneumonia strains.
The unique isolate of Salmonella enterica tested negative for the presence of the β-lactamase gene, confirming the sensitivity of this strain in the MIC assay, in contrast to its ampicillin resistance as determined by the KB method. The unique isolate E. fergusonii was sensitive to all the antibiotics, although the blaCTX-M-II gene was present according to PCR. Of the Enterobacter spp., represented by two gergoviae and two cloacae, one E. gergoviae was positive for blaSHV only, and one E. cloacae, for the blaTEM and blaDHA genes (Table 4).

2.7. Molecular Analysis for Virulence Factors in Enterobacteriaceae, Staphylococcus spp. and Clostridium perfringens (A to E)

All the E. coli (19 isolates) were analyzed for the presence of genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157, O26, O45, O103, O111, O121, and O145. The four virulence factors were also studied for all the other strains of Enterobacteriaceae (11 isolates). All the strains tested negative for the four virulent genes, except for one E. coli strain from dog id. 9, which carried the eae and the serogroup O111 genes (Table 4).
The Staphylococcus spp. strains showed negativity for the mecA and enterotoxin genes, except for S. xilosus (dog id. 4), which carried the enterotoxin D gene. One Clostridium perfringens strain (dog id. 3) showed the presence of the cpa gene, encoding the alpha-toxin.

2.8. Multidrug-Resistance Evaluation

To better assess the presence of multidrug-resistant strains among the 36 isolates tested using both methods (Kirby-Bauer and MIC), the results obtained with the two methods for the different antibiotic classes were compared. However, since the cards used to determine the MIC with VITEK® contain predetermined antibiotics, it was not possible to test the same molecules with both methods. For this reason, for the beta-lactam and tetracycline classes only, the comparison was based on different molecules of the same class, and for some other molecules (i.e., metronidazole), the comparison was not possible.
Among the Gram-negative strains, for the 27 Enterobacteriaceae, both methods confirmed sensitivity to the chloramphenicol class. Few variations were found between the two methods for penicillin and sulfonamides, while clearer differences emerged for cephalosporins, beta-lactams, aminoglycosides, fluoroquinolones, and tetracyclines (Table 5). The Pseudomonas aeruginosa strain was proven sensitive to all the antibiotic classes.
For the Gram-positives, no differences were found between the two methods for two of the eight Streptococcus spp., and the resistance of one strain to the tetracycline class was confirmed. For three of the eight Enterococcus spp., sensitivity to the chloramphenicol class was confirmed, but differences were evidenced for the fluoroquinolones and tetracyclines. For three of the eight Staphylococcus spp., both methods confirmed sensitivity to the aminoglycosides, chloramphenicol, and sulfonamides classes, and the resistance of only one strain to the lincosamides and fluoroquinolones. With MIC evaluation, the resistance of one strain of Staphylococcus spp. to macrolides was not confirmed, whereas resistance to the tetracycline class in one strain was observed.
Due to the variations among the methods, the results of the MIC method were considered to limit the overestimation of antimicrobial resistance. Bacterial strains showing resistance toward three or more antimicrobial classes were considered multidrug-resistant (MDR).
As a result of this comparison, 17 (47.2%) of the strains tested with both methods (15/27 Enterobacteriaceae, 1/2 Enterococcus faecium, and 1/1 Staphylococcus xylosus) were considered multidrug-resistant (Supplementary Material Tables S4 and S5). In particular, five strains of different bacterial species (1/16 E. coli, 1/4 Klebsiella pneumoniae, 1/2 Enterobacter cloacae, 1/2 Enterococcus faecium, and 1/1 Staphylococcus xylosus) showed resistance to three antibiotic classes; 1/16 strain of E. coli showed resistance to four antibiotics classes; seven strains (4/16 E. coli, 2/4 Klebsiella pneumoniae, and 1/2 Enterobacter cloacae), to five antibiotics classes; 2/16 strains of E. coli, to six antibiotics classes; and two strains (1/4 Klebsiella pneumoniae and 1/16 E. coli) showed resistance to seven and eight classes, respectively.
The multidrug-resistant strains were isolated from 13 dogs, and in one case, the presence of MDR was shown in all the strains isolated from the same dog. Indeed, the two strains, Klebsiella pneumoniae and E. coli, isolated from the stray dog id. 22 were resistant to seven (penicillin, cephalosporins, beta lactams, sulfonamides, fluoroquinolones, aminoglycosides, and tetracyclines) and five (penicillin, fluoroquinolones, aminoglycosides, chloramphenicol, and tetracyclines) classes, respectively. The same two bacterial species from dog id. 22 tested positive for the presence of the same four β-lactamase genes (Table 4). E. coli from dog id. 4 was resistant to five classes (penicillin, cephalosporins, beta lactams, sulfonamides, and tetracyclines), while Staphylococcus xylosus was resistant to three classes (tetracyclines, fluoroquinolones, and lincosamides); in the molecular analysis for the β-lactamase gene, the E. coli strain from dog 4 showed the presence of blaTEM and blaCTX-M-II genes (Table 4). In dog id. 10, the E. coli strain was resistant to eight antibiotic classes (penicillin, cephalosporins, beta lactams, sulfonamides, fluoroquinolones, aminoglycosides, tetracyclines, and chloramphenicol), although it was negative for the presence of β-lactamase genes when assessed by PCR, in contrast to the Klebsiella pneumoniae isolate, which was shown to be positive for the blaSHV gene and was resistant to three classes (penicillin, beta lactams, and tetracyclines). Multidrug-resistant strains of E. coli (the species isolated with the higher rate) were isolated from the intestines of three dogs and from the intestines and other organs of another five dogs.

3. Discussion

Despite the fact that vaccination has considerably reduced the occurrence, canine parvovirus infection remains a global threat to domestic and wild carnivores. Until now, studies have been focused on CPV infection and global spread, with limited studies on co-infections with bacteria or other viruses [17,20,33,34]. In this study, samples collected from dog carcasses with CPV infection were analyzed to evaluate the impact of the bacterial species, their susceptibility to antibiotics, and their multidrug resistance, along with other viral co-infections. In total, 18 dogs were strays, three were owned, one was housed in a city shelter, and one was just imported from an Eastern European country. The lack of any specific therapeutic treatment or previous vaccination for stray dogs and the potential stressful conditions for the others could have contributed to the fatal infection outcome.
The occurrence of CPV infection has been mainly reported in young dogs, probably related to the lack of specific and protective immunization or stressful conditions [5,35]. The vaccines currently used for CPV are safe and effective and cross-protect against all three variants [3,36,37]. Their rational use [38], together with appropriate sanitation procedures [39], still represents the most effective protective approach to preventing viral infection [40]. Moreover, vaccines could also reduce the unnecessary use of antimicrobials, as recently suggested [41,42].
The most commonly described pathological findings for CPV infection [5] were observed in all the carcasses during the anatomopathological examinations. However, other lesions not specifically associated with CPV were also observed. These anatomopathological observations might suggest a mixed pathological pattern, potentially due to bacterial co-infections, the effect of the bacterial toxins, or the systemic inflammatory response (SIRS) [14,43,44]. The data in this study suggest the need for more specific assays, particularly in the extra-intestinal organs from which bacteria were isolated, to assess their role and/or the roles of their toxins in determining pathological lesions. Additional studies are also necessary to evaluate CPV’s action in extra-intestinal sites, such as the central nervous system (CNS) or bone marrow.
Although in a few dogs (id. 6, 7, 11, 21, 22, 19) co-infections with other viruses (CCoV and CAdV-1) were assessed, the results confirm that CPV infection remained the main cause of viral enteritis and acute hemorrhagic diarrhea syndrome (AHDS) in young dogs [33,34]. Therefore, a diagnostic panel for the main pathogenic bacteria and multiple viral agents should be considered in dogs with suspected infectious gastrointestinal disease [21,34].
From all the samples, forty-three bacterial strains were isolated, with a prevalence of Gram-negative groups and, particularly, E. coli species, isolated mainly from the organs of the gastrointestinal tract (liver, spleen, and intestine) and, less frequently, from extraintestinal organs. Furthermore, few species belonging to the Gram-positive group were isolated. Bacterial isolation from intestinal tissues was performed in five dogs only, while, in the others, bacteria were isolated mainly from the brain but also other tissues, suggesting a systemic or multi-organ infection. The evidence in this study of toxin-producing bacteria, such as Clostridium perfringens, and of other pathogenic bacterial strains in the brain suggests their role in the development of the neurological clinical signs, such as depression, commonly observed in live dogs with CPV infection [5,14,45]. However, further studies on the potential association of CPV with neurological lesions, as suggested in other reports [34,46,47,48,49], are necessary.
According to the results, the risk some bacteria pose of fatal outcomes in dogs with CPV infection appears to be partially limited. Indeed, most of the isolated bacteria (E. coli strains) represented normal intestinal flora and the fact that their presence was restricted to the enteric tract confirms their limited role in the pathogenesis of the dogs. Therefore, antibiotic therapy would most likely not have been necessary in these cases. Conversely, in eleven dogs, E. coli was isolated from extra-enteric organs, and in two dogs, bacteria harboring toxin genes (Clostridium perfringens and Staphylococcus xylosus) were also evidenced. In almost half of the analyzed canine carcasses, two or three different bacterial species were isolated, suggesting their potential role in developing the clinical signs and contributing to the exitus, or the observed pathological findings, as previously suggested [21,50]. In these cases, antimicrobial therapy in vivo might have been suggested and could have been effective. However, the accurate evaluation of the clinical evidence of sepsis status and antimicrobial resistance is important before considering any empirical therapy in order to avoid unnecessary treatment which could favor the spread of antimicrobial-resistant strains.
Despite the potential marginal role of the bacteria in the clinical outcome, this study evidenced the presence of multidrug-resistant bacteria in dogs with parvovirosis. In some cases, the isolates showed resistance to the most important antimicrobial drugs in human medicine, such as second- and fourth-generation cephalosporins, macrolides, lincosamides, nitroimidazoles, and glycopeptides while all the Gram-positive bacteria were resistant to fourth-generation cephalosporins and, with the exception of two Clostridium perfringens strains, also to nitroimidazoles. A slightly higher sensitivity to the ceftriaxone over cefovecin (third gen. cephalosporins) was also observed.
The analysis of the β-lactamase genes of Enterobacteriaceae showed that 15/30 of the strains harbored one to four genes of resistance. In one E. coli and one Klebsiella pneumoniae from dog id. 22, four genes of resistance (blaTEM, blaSHV, blaOXA, and blaCTX-M-II) were detected. Although the gene blaSHV is commonly present in most K. pneumoniae strains at chromosomal locations, the same combination of different genes present in different bacterial strains in the same dog suggests acquisition through genetic horizontal transfer. Another K. pneumoniae derived from dog id. 12 carried the same four genes. The evidence of these bacteria in the enteric tract suggests their potential shedding via feces, and this deserves attention considering their potential zoonotic role and the possibility of spreading to humans. These strains also showed the highest resistance in both the Kirby–Bauer and MIC assays.
In the treatment of dogs with parvoviral enteritis there is no specific therapy, only supportive care approaches. Although some reports warn about the potential risks connected to the use of antibiotics [14,43], an intravenous or subcutaneous broad spectrum of bactericidal antibiotics is commonly used in addition to therapy. Penicillin, alone or in combination with beta-lactamase inhibitors, cephalosporins, fluoroquinolones, metronidazole, and aminoglycosides, is the most commonly used antibiotics that have been reported [15,51,52]. To date, official data on the use or sale of antimicrobials in Italy for the treatment of gastrointestinal infections in dogs are not available. Nonetheless, a guideline for the prudent use of antibiotics in companion animals has been provided [53] along with surveys and cross-sectional studies describing the use of antimicrobials for companion animals [54,55,56]. Similar studies involving other European countries, including Italy, have recently been published [57,58]. Moreover, most recent updates of canine parvoviral enteritis recommend the use of broad-spectrum antibiotics such as ampicillins, cephalosporins, nitroimidazoles, or fluoroquinolones [59]. Although a national survey specifically on the use of antimicrobials in dogs with CPV infection is not available, all of these studies, as well as the guidelines, cite specific antibiotics (i.e., nitroimidazoles such as metronidazole, alone or in addition with spiramycin or cephalosporins) and CIAs (i.e., fluoroquinolones or third-generation cephalosporins) for common use in the generically defined gastrointestinal disease. Since 2017, computerized prescriptions for veterinary medical products (defined as Ricetta Elettronica Veterinaria, REV) have been available in Italy, replacing paper prescriptions for antimicrobials under the direct control of the Ministry of Health. Data analysis could contribute to the categorization of the antimicrobials used for companion animals, supporting future strategies to combat AMR, along with the increasing attention being paid to multi-resistant pathogens found in companion animals that are harmful for humans.
This study outlines the high bacterial resistance to some of the antibiotics commonly used in the treatment of parvoviral enteritis, such as third- and fourth-generation cephalosporins and metronidazole, which pose a high risk of the spread of resistance to antibiotics that are very important for human health. Moreover, inappropriate and ineffective empirical treatments of CPV infection, such as intravenous therapy with narrow-spectrum antimicrobials, potentially contribute to the occurrence of other short- or long-term effects, such as damage to fecal microbiota, neurotoxicity, and chronic gastrointestinal disease [60,61,62]. Given this evidence, the real need for antibiotic therapy and its benefits should be assessed.
The rational rather than empirical use of antibiotics could contribute to the effective control of antimicrobial resistance. The concerns related to AMR are increasing, especially for those involving important antimicrobial classes, such as the third- or higher-generation cephalosporins, glycopeptides, macrolides, ketolides, polymyxins, and quinolones included in the lists of international health institutions [24]. Due to the threat posed to human health, the guidelines on the rational use of antibiotics mainly refer to food-producing animals or animal production practices and the role of companion animals is neglected [63,64]. Moreover, the need to elucidate the role of companion animals in the spread of antibiotic resistance is highlighted by the fact that some of the microorganisms included in the WHO’s list of globally prioritized antibiotic-resistant bacteria [65] are often isolated from companion animals. In our study, four strains of Klebsiella pneumoniae, representing a threat to human health in hospital settings [66], showed more than one β-lactamase gene and multidrug resistance. An E. coli strain isolated from a young stray dog showed the O111 serogroup and eae genes. The O111 serotype and enteroaggregative intimin (eae) genes related to E. coli strains are responsible for diarrhea problems in children [67]. Moreover, four E. coli, one Escherichia fergusonii, two Klebsiella pneumoniae, and one Enterobacter cloacae were shown to be resistant to antibiotics considered a last line of defense against resistant infections such as colistin sulphate, imipenem, chloramphenicol, and ceftriaxone. We cannot rule out the possibility that resistant bacterial strains were transferred from humans to animals, since some of the tested puppies might have been abandoned by owners that could not keep the newborn animals. The presence of multidrug resistance could be related to the household environment and it is possible that, for pets with close relations to humans, AMR originates from human sources thus confirming the importance of the One Health approach. Moreover, less common bacteria indicated as potential agents of zoonoses were also isolated: Salmonella enterica subsp. enterica serovar Schleissheim [68], E. fergusonii [69], Str. pseudoporcinus [70], and Str. canis [71]. This evidence suggests a potential risk for humans connected to the shedding of zoonotic bacteria species carrying drug resistance. Due to this evidence being found in stray and shelter-housed dogs, the roles of these species should be assessed and considered as part of sanitation protocols to limit the contamination of shelters and veterinary clinics, thus limiting the risks posed to the personnel of shelters and veterinarians.
Some limitations—particularly the low availability of tissue samples from dead dogs naturally infected with canine parvovirus—prevent in-depth data analysis. First, the lack of negative controls is a potential limitation: since this was a descriptive study, intended only to evaluate the antimicrobial susceptibility and multidrug-resistance profiles of bacterial species derived from tissue samples of dogs with canine parvovirus infection, samples from CPV-negative dogs were considered non-ideal as negative controls.
As this observational study was based only on samples collected for routine diagnostic purposes, aiming to describe and highlight the presence of multi-resistant bacteria in these targeted individuals, no negative controls were defined.
Another limit was related to the lack of specific anamnestic and clinical information, particularly on the use of antimicrobials for therapies, which prevents speculation on the meaning of the resistance found in the analyzed strains. Therefore, in light of these limitations, further studies are necessary in order to derive in-depth deductions.
Antibiotic treatment is sometimes used in canine parvoviral infection but, as shown in this study, the evidence of multi-resistant bacteria with potential for intra- or cross-species transmission should be carefully considered before unnecessary antimicrobial treatments are undertaken. Dogs, as companion animals, are usually reared and housed in close contact with humans [72,73] and, therefore, a One Health perspective is imperative for global public health.

4. Materials and Methods

4.1. Clinical Samples

Tissue samples from 23 dead dogs suspected of having parvovirosis were analyzed. Samples were collected from May 2018 to October 2019 and analyzed for diagnostic purposes. Carcasses had already been submitted by public and private veterinary practitioners to ascertain the causa mortis. Most of these subjects were stray dogs (n = 18) and the others were owned dogs (n = 3), shelter dogs (n = 1) and imported dogs (n = 1). The veterinary public services recovered all but one of the roaming strays showing clinical gastroenteric signs, all of which died just after admittance; the other died just before it could be recovered. Other carcasses were submitted by private or public veterinary practitioners with clinical suspicion of infectious gastrointestinal disease in almost all of them. No other anamnestic or clinical information was provided, including vaccination statuses or therapies. The carcasses were subjected to necropsy after admittance or storage at −20 °C. During necropsy, tissue samples (brain, lungs, heart, spleen, liver, intestine, mesenteric lymph nodes, and kidneys) were collected, stored at −20 °C, and subjected to virological and bacteriological assays. The details are summarized in Supplementary Materials Table S1.

4.2. Parvovirus PCR and Molecular Characterization of CPV Strains

Organ homogenates were obtained as previously described [74]. DNA was extracted from homogenates using the DNeasy Blood & Tissue Kit (Qiagen S.p.A., Hilden, Germany), according to the manufacturer’s instructions. The presence of CPV DNA was confirmed using a primer pair [75] in a PCR protocol amplifying a 700 bp fragment of the VP2 gene, as previously described [28]. Briefly, PCR was carried out using the GoTaq G2 DNA Polymerase (Promega Italia s.r.l., Milan, Italy) in a 50 μL reaction mix consisting of 10 µL of 5× GoTaq® Reaction Buffer, 1 µL of MgCl2 (25 mM), 1 µL of dNTP mix (10 mM), 0.5 µL of each primer VP2-850-Forward and VP2-1550-Reverse (0.5 µM), 0.25 µL of GoTaq® G2 DNA Polymerase, 31.75 μL of nuclease-free water, and 5 μL of DNA extract. Amplification was conducted under the following thermal conditions: 94 °C for 2 min to activate TaqPol followed by 40 cycles of 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min as well as a final extension of 72 °C for 10 min.
The nearly complete VP2 gene sequence (a 1745-bp fragment) was assessed using a primer pair [76] and direct sequencing [77].
Sequencing encompassing both CPV ORFs (including NS and VP genes) was carried out using the primer pairs developed by Pérez et al. [78], as previously described, and the amplicons were directly sequenced using forward, reverse, and internal primers [29].
The nucleotide VP2 coding sequences were obtained using the ClustalW program and analyzed using the BioEdit software. The sequences were submitted to nBLAST to search related sequences in public domain databases. The CPV antigenic variants (CPV-2a, 2b and 2c) were deduced based on the 426-VP2 amino acid residue [79].
To elucidate the genetic relationships between the obtained CPV strains and the dataset of sequences obtained from the NCBI database, a phylogenetic tree was constructed with the MEGA-X software, using the maximum-likelihood (ML) method according to the Tamura 3-parameter (T92) model with discrete Gamma distribution (+G) employing five rate categories, assuming that a certain fraction of the sites were evolutionarily invariable (+I), and employing bootstrap analyses with 1000 replicates. The phylogeny is depicted in Supplementary Materials Figure S2, showing a representative CPV strain for each genetic and antigenic variant.
These sequence data have been previously or newly submitted to the DDBJ/EMBL/GenBank databases under accession numbers reported in Supplementary Materials Table S1.

4.3. Additional Virologic Tests

RNA was extracted from samples using the QIAamp Viral RNA Mini Kit (Qiagen S.p.A., Hilden, Germany), according to the manufacturer’s instructions. The extracted DNA and RNA were amplified using a set of gel-based or real-time (RT) PCR assays useful for the detection of CDV [80], CAdVs types 1 and 2 [81], CCoV [82], and CRoV [83]. The details are summarized in Supplementary Materials Table S6.

4.4. Bacterial Isolation

For the tissue samples collected from all the dogs, bacterial isolation was performed using selective and differential agar (MacConkey agar, Columbia blood agar and Mannitol Salt agar) incubated at 37 °C for 24 h. Moreover, Columbia blood agar plates were anaerobically incubated with the AnaeroGen™ Anaerobic System (Oxoid, Milano, Italy) to isolate anaerobic bacteria.
For the Salmonella spp. culture, pre-enrichment in Buffered Peptone water was performed, followed by two enrichments in Selenite Cystine (SC) and Rappaport-Vassiliadis (RV) broths, and incubated, respectively, at 37 °C and 42 °C for 24 h. The enrichment broths were then plated on Xylose–Lysine Deoxycholate Agar (XLD) and Brilliant Green Agar (BGA).
The identification of the isolated strains was carried out with the biochemical API® system and Vitek® 2 system (bioMérieux, Craponne, France). For the Salmonella spp. strains, after identification by API20E®, serological typing was performed.

4.5. Disk Diffusion Method

The antimicrobial susceptibility of the bacterial strains isolated (n = 43) was evaluated by the disk diffusion method (Kirby-Bauer) on Mueller-Hinton agar, according to the guidelines of the Clinical and Laboratory Standards Institute [84]. A standard panel of 22 antibiotics was used: amoxicillin-clavulanic acid (AMC), 30 μg; ampicillin (AMP), 10 μg; aztreonam (ATM), 30 μg; cefadroxil (CFR), 30 μg; cephalexin (CL), 30 μg; cefovecin (CVN), 30 μg; ceftriaxone (CRO), 30 μg; cefquinome (CEQ), 30 μg; cefuroxime (CXM), 30 μg; clindamycin (DA), 2 μg; chloramphenicol (C), 30 μg; colistin sulfate (CT), 10 μg; doxycycline (DO), 30 μg; enrofloxacin (ENR), 5 μg; gentamicin (CN), 10 μg; imipenem (IPM), 10 μg; marbofloxacin (MAR), 5 μg; methicillin (MET), 5 μg; metronidazole (MPZ), 4 μg; spiramycin (SP), 100 μg; sulfamethoxazole/trimethoprim (SXT), 23.75 μg/1.25 μg; vancomycin (VA), 30 μg. The sensitivity model was evaluated by measuring the diameter of the inhibition zone, and isolates were considered resistant, intermediate, or susceptible according to the CLSI ranges [84].

4.6. Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentrations (MICs) of 36 of the isolated strains were determined with the Vitek® 2 system (bioMérieux, Craponne, France), with specific panels of antibiotics selected according to the identified species. The VITEK® AST-GN65 card was used to determine the susceptibility of 28 strains of isolated Gram-negative aerobic bacilli, while the VITEK® AST-GP81 card was used to determine the susceptibility of three Enterococcus spp. And three Staphylococcus spp. The VITEK® AST-ST03 card was used for the two Streptococcus spp., whose MIC values were expressed in µg/mL. According to the breakpoints expressed in vet CLSI 2017 v8.02 and CLSI M100-S25 (2015) [84,85], the isolates were categorized as resistant, intermediate, or susceptible.

4.7. Detection of β-Lactamase Genes

Two multiplex PCRs were performed to amplify the β-lactamase genes in the Enterobacteriaceae isolates as described by Kim et al. [86]. The first multiplex assay (named Set I) was designed to detect the blaTEM, blaSHV, and blaCTX-M-IV group- (8–10) and blaOXA β-lactamase-encoding genes, and the second assay (named Set II) was designed to detect blaCTX-M-I group-, blaCTX-M-II group-, blaCMY-II-, and blaDHA-encoding genes. The DNA amplifications were carried out in the GeneAmp™ PCR System 2700 thermal cycle (Applied Biosystems, Foster City, CA, USA). Both assays used identical cycling conditions: the thermal cycling profile consisted of an initial denaturation at 95 °C for 15 min, followed by 30 cycles of 94 °C for 30 s, 61 °C for 90 s, and 72 °C for 90 s and a final extension at 72 °C for 10 min. The sizes of the PCR products were analyzed by electrophoresis on a 2% agarose gel containing GelRed® (Biotium, San Francisco, CA, USA) (4 µL per 100 mL) in 0.5× TBE at 100 V for 1 h, and visualized using GeneSys (Syngene, Cambridge, UK).

4.8. Detection of Genes for Toxins of Staphylococcus spp. and of Clostridium perfringens (A to E)

Two multiplex PCR assays were used to amplify the sea-see and tsst-1, eta, etb, mecA (Set I), and seg, seh, sei, sej, and sep (Set II) genes for toxins of Staphylococcus spp. as described by Vitale et al. [87].
A multiplex PCR assay was used to detect the toxin genes cpa, cpb, etx, iap, cpe, and cpb2 of Clostridium perfringens, according to the method described by Baums et al. [88]. The PCR results were visualized by electrophoresis on a 1.5% agarose gel containing GelRed® (Biotium, San Francisco, CA, USA) (4 µL per 100 mL) in 0.5× TBE at 100 V for 1 h and visualized using GeneSys (Syngene, Cambridge, UK).

4.9. Serogroup Identification in E. coli and Virulent Genes’ Identification in Enterobacteriaceae

A multiplex polymerase chain reaction (mPCR) was used to detect the 11 genes that encode serogroup-specific O-antigens and four major virulence factors (eae–intimin adherence protein, enterohemorrhagic hemolysin A (EHEC hlyA), and Shiga toxins [Stx] 1 and 2) so as to detect O157 and the “top six” non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC) as described by Bai et al. [89]. The search for genes coding for the four virulence factors mentioned above was conducted on all the strains of Enterobacteriaceae isolated.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/antibiotics11020142/s1. Figure S1: Gross lesions observed at necropsy, Figure S2: Maximum likelihood tree based on 158 full-length VP2 gene sequences of canine parvovirus type 2 strains, Table S1: Details of collected and tested samples, Table S2: Antibiotic sensitivity results according to minimum inhibitory concentration (MIC) method for the Gram-negative strains (n = 28), Table S3: Antibiotic sensitivity results with minimum inhibitory concentration (MIC) method for the Gram-positive strains (n = 8), Table S4: Comparison of the results obtained with the two methods for MDR Enterobacteriaceae strains (n = 15), Table S5: Comparison of the results obtained with the two methods for MDR Gram-positive strains (n = 2), Table S6: Details on additional virologic tests.

Author Contributions

Conceptualization, G.S., D.G. and F.M.; methodology, G.S., D.G., M.V. and F.A.; formal analysis, G.S., D.G., F.M., M.V. and F.A.; investigation, G.S., D.G., F.L., G.C., M.L.G. and V.R.; resources, G.P., M.V., F.A. and D.V.; data curation, G.S., D.G., F.M. and M.V.; writing—original draft preparation, G.S., D.G., F.M. and M.V.; writing—review and editing, G.S., D.G., F.M., M.V. and D.V.; visualization, G.S. and F.M.; supervision, M.V., A.G., G.P., F.A. and D.V.; project administration, F.M., A.G. and D.V.; funding acquisition, A.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministero della Salute (Italy), Ricerca Corrente IZS SI 03/18 RC “Studio del potenziale zoonosico e caratterizzazione genomica dei virus enterici del cane”.

Institutional Review Board Statement

Ethical review and approval were not required for this study since no experiment on live animals was performed; the study was carried out using tissue samples collected from dead animals submitted at the Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri” for diagnostic purposes.

Informed Consent Statement

Not applicable.

Data Availability Statement

Sequence data were submitted to the DDBJ/EMBL/GenBank databases under accession numbers MT981020–MT981039.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Carmichael, L.E. An annotated historical account of canine parvovirus. J. Vet. Med. B Infect. Dis. Vet. Public Health 2005, 52, 303–311. [Google Scholar] [CrossRef] [PubMed]
  2. Pratelli, A.; Cavalli, A.; Martella, V.; Tempesta, M.; Decaro, N.; Carmichael, L.E.; Buonavoglia, C. Canine parvovirus (CPV) vaccination: Comparison of neutralizing antibody responses in pups after inoculation with CPV2 or CPV2b modified live virus vaccine. Clin. Diagn. Lab. Immunol. 2001, 8, 612–615. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Spibey, N.; Greenwood, N.M.; Sutton, D.; Chalmers, W.S.K.; Tarpey, I. Canine parvovirus type 2 vaccine protects against virulent challenge with type 2c virus. Vet. Microbiol. 2008, 128, 48–55. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Voorhees, I.E.H.; Lee, H.; Allison, A.B.; Lopez-Astacio, R.; Goodman, L.B.; Oyesola, O.O.; Omobowale, O.; Fagbohun, O.; Dubovi, E.J.; Hafenstein, S.L.; et al. Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine parvovirus Host Adaptation and Spread. J. Virol. 2019, 94, e01162-19. [Google Scholar] [CrossRef]
  5. Decaro, N.; Buonavoglia, C. Canine parvovirus—A review of epidemiological and diagnostic aspects, with emphasis on type 2c. Vet. Microbiol. 2012, 155, 1–12. [Google Scholar] [CrossRef]
  6. Cotmore, S.F.; Agbandje-McKenna, M.; Canuti, M.; Chiorini, J.A.; Eis-Hubinger, A.-M.; Hughes, J.; Mietzsch, M.; Modha, S.; Ogliastro, M.; Pénzes, J.J.; et al. ICTV Virus Taxonomy Profile: Parvoviridae. J. Gen. Virol. 2019, 100, 367–368. [Google Scholar] [CrossRef]
  7. Cotmore, S.F.; Agbandje-McKenna, M.; Chiorini, J.A.; Mukha, D.V.; Pintel, D.J.; Qiu, J.; Soderlund-Venermo, M.; Tattersall, P.; Tijssen, P.; Gatherer, D.; et al. The family Parvoviridae. Arch. Virol. 2014, 159, 1239–1247. [Google Scholar] [CrossRef]
  8. Parrish, C.R.; O’Connell, P.H.; Evermann, J.F.; Carmichael, L.E. Natural variation of canine parvovirus. Science 1985, 230, 1046–1048. [Google Scholar] [CrossRef]
  9. Parrish, C.R.; Aquadro, C.F.; Strassheim, M.L.; Evermann, J.F.; Sgro, J.Y.; Mohammed, H.O. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J. Virol. 1991, 65, 6544–6552. [Google Scholar] [CrossRef] [Green Version]
  10. Buonavoglia, C.; Martella, V.; Pratelli, A.; Tempesta, M.; Cavalli, A.; Buonavoglia, D.; Bozzo, G.; Elia, G.; Decaro, N.; Carmichael, L. Evidence for evolution of Canine parvovirus type 2 in Italy. J. Gen. Virol. 2001, 82, 3021–3025. [Google Scholar] [CrossRef]
  11. Mohr, A.J.; Leisewitz, A.L.; Jacobson, L.S.; Steiner, J.M.; Ruaux, C.G.; Williams, D.A. Effect of early enteral nutrition on intestinal permeability, intestinal protein loss, and outcome in dogs with severe parvoviral enteritis. J. Vet. Intern. Med. 2003, 17, 791–798. [Google Scholar] [CrossRef] [PubMed]
  12. Armenise, A.; Trerotoli, P.; Cirone, F.; De Nitto, A.; De Sario, C.; Bertazzolo, W.; Pratelli, A.; Decaro, N. Use of recombinant canine granulocyte-colony stimulating factor to increase leukocyte count in dogs naturally infected by canine parvovirus. Vet. Microbiol. 2019, 231, 177–182. [Google Scholar] [CrossRef] [PubMed]
  13. Gerlach, M.; Proksch, A.-L.; Dörfelt, R.; Unterer, S.; Hartmann, K. Therapy of Canine parvovirus infection—Review and current insights. Tierarztl. Prax. Ausg. K Kleintiere Heimtiere 2020, 48, 26–37. [Google Scholar] [CrossRef] [PubMed]
  14. Goddard, A.; Leisewitz, A.L. Canine parvovirus. Vet. Clin. N. Am. Small Anim. Pract. 2010, 40, 1041–1053. [Google Scholar] [CrossRef] [PubMed]
  15. Pereira, G.Q.; Gomes, L.A.; Santos, I.S.; Alfieri, A.F.; Weese, J.S.; Costa, M.C. Fecal microbiota transplantation in puppies with Canine parvovirus infection. J. Vet. Intern. Med. 2018, 32, 707–711. [Google Scholar] [CrossRef] [PubMed]
  16. Park, J.S.; Guevarra, R.B.; Kim, B.-R.; Lee, J.H.; Lee, S.H.; Cho, J.H.; Kim, H.; Cho, J.H.; Song, M.; Lee, J.-H.; et al. Intestinal Microbial Dysbiosis in Beagles Naturally Infected with Canine Parvovirus. J. Microbiol. Biotechnol. 2019, 29, 1391–1400. [Google Scholar] [CrossRef] [PubMed]
  17. Zheng, Y.; Hao, X.; Lin, X.; Zheng, Q.; Zhang, W.; Zhou, P.; Li, S. Bacterial diversity in the feces of dogs with CPV infection. Microb. Pathog. 2018, 121, 70–76. [Google Scholar] [CrossRef]
  18. Otto, C.M.; Drobatz, K.J.; Soter, C. Endotoxemia and tumor necrosis factor activity in dogs with naturally occurring parvoviral enteritis. J. Vet. Intern. Med. 1997, 11, 65–70. [Google Scholar] [CrossRef]
  19. Botha, W.J.; Schoeman, J.P.; Marks, S.L.; Whitehead, Z.; Annandale, C.H. Prevalence of Salmonella in juvenile dogs affected with parvoviral enteritis. J. S. Afr. Vet. Assoc. 2018, 89, e1–e6. [Google Scholar] [CrossRef] [Green Version]
  20. Silva, R.O.S.; Dorella, F.A.; Figueiredo, H.C.P.; Costa, É.A.; Pelicia, V.; Ribeiro, B.L.D.; Ribeiro, M.G.; Paes, A.C.; Megid, J.; Lobato, F.C.F. Clostridium perfringens and C. difficile in parvovirus-positive dogs. Anaerobe 2017, 48, 66–69. [Google Scholar] [CrossRef] [Green Version]
  21. Duijvestijn, M.; Mughini-Gras, L.; Schuurman, N.; Schijf, W.; Wagenaar, J.A.; Egberink, H. Enteropathogen infections in canine puppies: (Co-)occurrence, clinical relevance and risk factors. Vet. Microbiol. 2016, 195, 115–122. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Da Rocha Gizzi, A.B.; Oliveira, S.T.; Leutenegger, C.M.; Estrada, M.; Kozemjakin, D.A.; Stedile, R.; Marcondes, M.; Biondo, A.W. Presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel. BMC Vet. Res. 2014, 10, 23. [Google Scholar] [CrossRef] [Green Version]
  23. McEwen, S.A.; Collignon, P.J. Antimicrobial Resistance: A One Health Perspective. Microbiol. Spectr. 2018, 6, ARBA-0009-2017. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. WHO (World Health Organization). Critically Important Antimicrobials for Human Medicine, 6th ed.; WHO Press: Geneva, Switzerland, 2019; Available online: https://apps.who.int/iris/bitstream/handle/10665/312266/9789241515528-eng.pdf?ua=1 (accessed on 7 October 2020).
  25. Collignon, P.J.; McEwen, S.A. One Health-Its Importance in Helping to Better Control Antimicrobial Resistance. Trop. Med. Infect. Dis. 2019, 4, 22. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Guardabassi, L.; Schwarz, S.; Lloyd, D.H. Pet animals as reservoirs of antimicrobial-resistant bacteria. J. Antimicrob. Chemother. 2004, 54, 321–332. [Google Scholar] [CrossRef]
  27. Decaro, N.; Martella, V.; Elia, G.; Desario, C.; Campolo, M.; Lorusso, E.; Colaianni, M.L.; Lorusso, A.; Buonavoglia, C. Tissue distribution of the antigenic variants of Canine parvovirus type 2 in dogs. Vet. Microbiol. 2007, 121, 39–44. [Google Scholar] [CrossRef]
  28. Mira, F.; Dowgier, G.; Purpari, G.; Vicari, D.; Di Bella, S.; Macaluso, G.; Gucciardi, F.; Randazzo, V.; Decaro, N.; Guercio, A. Molecular typing of a novel Canine parvovirus type 2a mutant circulating in Italy. Infect. Genet. Evol. 2018, 61, 67–73. [Google Scholar] [CrossRef]
  29. Mira, F.; Canuti, M.; Purpari, G.; Cannella, V.; Di Bella, S.; Occhiogrosso, L.; Schirò, G.; Chiaramonte, G.; Barreca, S.; Pisano, P.; et al. Molecular Characterization and Evolutionary Analyses of Carnivore Protoparvovirus 1 NS1 Gene. Viruses 2019, 11, 308. [Google Scholar] [CrossRef] [Green Version]
  30. Mira, F.; Purpari, G.; Lorusso, E.; Di Bella, S.; Gucciardi, F.; Desario, C.; Macaluso, G.; Decaro, N.; Guercio, A. Introduction of Asian Canine parvovirus in Europe through dog importation. Transbound. Emerg. Dis. 2018, 65, 16–21. [Google Scholar] [CrossRef]
  31. Mira, F.; Purpari, G.; Di Bella, S.; Colaianni, M.L.; Schirò, G.; Chiaramonte, G.; Gucciardi, F.; Pisano, P.; Lastra, A.; Decaro, N.; et al. Spreading of Canine parvovirus type 2c mutants of Asian origin in southern Italy. Transbound. Emerg. Dis. 2019, 66, 2297–2304. [Google Scholar] [CrossRef]
  32. Leclercq, R.; Cantón, R.; Brown, D.F.J.; Giske, C.G.; Heisig, P.; MacGowan, A.P.; Mouton, J.W.; Nordmann, P.; Rodloff, A.C.; Rossolini, G.M.; et al. EUCAST expert rules in antimicrobial susceptibility testing. Clin. Microbiol. Infect. 2013, 19, 141–160. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Cardillo, L.; Piegari, G.; Iovane, V.; Viscardi, M.; Alfano, F.; Cerrone, A.; Pagnini, U.; Montagnaro, S.; Galiero, G.; Pisanelli, G.; et al. Lifestyle as Risk Factor for Infectious Causes of Death in Young Dogs: A Retrospective Study in Southern Italy (2015–2017). Vet. Med. Int. 2020, 2020, 6207297. [Google Scholar] [CrossRef] [PubMed]
  34. Dowgier, G.; Lorusso, E.; Decaro, N.; Desario, C.; Mari, V.; Lucente, M.S.; Lanave, G.; Buonavoglia, C.; Elia, G. A molecular survey for selected viral enteropathogens revealed a limited role of Canine circovirus in the development of canine acute gastroenteritis. Vet. Microbiol. 2017, 204, 54–58. [Google Scholar] [CrossRef] [PubMed]
  35. Greene, C.E.; Decaro, N. Canine Viral Enteritis. In Infectious Diseases of Dog and Cat, 4th ed.; Greene, C.E., Ed.; Elsevier Saunders: Philadelphia, PA, USA, 2012; pp. 67–80. [Google Scholar]
  36. Wilson, S.; Illambas, J.; Siedek, E.; Stirling, C.; Thomas, A.; Plevová, E.; Sture, G.; Salt, J. Vaccination of dogs with canine parvovirus type 2b (CPV-2b) induces neutralising antibody responses to CPV-2a and CPV-2c. Vaccine 2014, 32, 5420–5424. [Google Scholar] [CrossRef] [PubMed]
  37. Larson, L.J.; Schultz, R.D. Do two current Canine parvovirus type 2 and 2b vaccines provide protection against the new type 2c variant? Vet. Ther. 2008, 9, 94–101. [Google Scholar] [PubMed]
  38. Day, M.J.; Horzinek, M.C.; Schultz, R.D.; Squires, R.A.; Vaccination Guidelines Group (VGG) of the World Small Animal Veterinary Association (WSAVA). WSAVA Guidelines for the vaccination of dogs and cats. J. Small Anim. Pract. 2016, 57, E1–E45. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Cavalli, A.; Marinaro, M.; Desario, C.; Corrente, M.; Camero, M.; Buonavoglia, C. In vitro virucidal activity of sodium hypochlorite against Canine parvovirus type 2. Epidemiol. Infect. 2018, 146, 2010–2013. [Google Scholar] [CrossRef] [Green Version]
  40. Decaro, N.; Buonavoglia, C.; Barrs, V.R. Canine parvovirus vaccination and immunisation failures: Are we far from disease eradication? Vet. Microbiol. 2020, 247, 108760. [Google Scholar] [CrossRef]
  41. Buchy, P.; Ascioglu, S.; Buisson, Y.; Datta, S.; Nissen, M.; Tambyah, P.A.; Vong, S. Impact of vaccines on antimicrobial resistance. Int. J. Infect. Dis. 2020, 90, 188–196. [Google Scholar] [CrossRef] [Green Version]
  42. Bloom, D.E.; Black, S.; Salisbury, D.; Rappuoli, R. Antimicrobial resistance and the role of vaccines. Proc. Natl. Acad. Sci. USA 2018, 115, 12868–12871. [Google Scholar] [CrossRef] [Green Version]
  43. Prittie, J. Canine parvoviral enteritis: A review of diagnosis, management, and prevention. J. Vet. Emerg. Crit. Car. 2004, 14, 167–176. [Google Scholar] [CrossRef]
  44. Alves, F.; Prata, S.; Nunes, T.; Gomes, J.; Aguiar, S.; Aires da Silva, F.; Tavares, L.; Almeida, V.; Gil, S. Canine parvovirus: A predicting canine model for sepsis. BMC Vet. Res. 2020, 16, 199. [Google Scholar] [CrossRef] [PubMed]
  45. Miranda, C.; Carvalheira, J.; Parrish, C.R.; Thompson, G. Factors affecting the occurrence of Canine parvovirus in dogs. Vet. Microbiol. 2015, 180, 59–64. [Google Scholar] [CrossRef] [PubMed]
  46. Marenzoni, M.L.; Calò, P.; Foiani, G.; Tossici, S.; Passantino, G.; Decaro, N.; Mandara, M.T. Porencephaly and Periventricular Encephalitis in a 4-month-old Puppy: Detection of Canine parvovirus Type 2 and Potential Role in Brain Lesions. J. Comp. Pathol. 2019, 169, 20–24. [Google Scholar] [CrossRef]
  47. Zhao, Y.; Lin, Y.; Zeng, X.; Lu, C.; Hou, J. Genotyping and pathobiologic characterization of Canine parvovirus circulating in Nanjing, China. Virol. J. 2013, 10, 272. [Google Scholar] [CrossRef] [Green Version]
  48. Schaudien, D.; Polizopoulou, Z.; Koutinas, A.; Schwab, S.; Porombka, D.; Baumgärtner, W.; Herden, C. Leukoencephalopathy associated with parvovirus infection in Cretan hound puppies. J. Clin. Microbiol. 2010, 48, 3169–3175. [Google Scholar] [CrossRef] [Green Version]
  49. Schwab, S.; Herden, C.; Seeliger, F.; Papaioannou, N.; Psalla, D.; Polizopulou, Z.; Baumgärtner, W. Non-suppurative meningoencephalitis of unknown origin in cats and dogs: An immunohistochemical study. J. Comp. Pathol. 2007, 136, 96–110. [Google Scholar] [CrossRef]
  50. Schoeman, J.P.; Goddard, A.; Leisewitz, A.L. Biomarkers in Canine parvovirus enteritis. N. Z. Vet. J. 2013, 61, 217–222. [Google Scholar] [CrossRef] [Green Version]
  51. Venn, E.C.; Preisner, K.; Boscan, P.L.; Twedt, D.C.; Sullivan, L.A. Evaluation of an outpatient protocol in the treatment of canine parvoviral enteritis. J. Vet. Emerg. Crit. Care 2017, 27, 52–65. [Google Scholar] [CrossRef] [Green Version]
  52. Mylonakis, M.E.; Kalli, I.; Rallis, T.S. Canine parvoviral enteritis: An update on the clinical diagnosis, treatment, and prevention. Vet. Med. Auckl 2016, 7, 91–100. [Google Scholar] [CrossRef] [Green Version]
  53. Barbarossa, A.; Casadio, C.; Diegoli, G.; Fontana, M.C.; Giunti, M.; Miraglia, V.; Rambaldi, J.; Rubini, M.; Torresani, G.; Trambajolo, G.; et al. LINEE GUIDA Uso Prudente Dell’antibiotico Negli Animali da Compagnia. 2018. Available online: https://www.alimenti-salute.it/sites/default/files/Linee%20Guida%20PETs%202018.pdf (accessed on 22 November 2021).
  54. Escher, M.; Vanni, M.; Intorre, L.; Caprioli, A.; Tognetti, R.; Scavia, G. Use of antimicrobials in companion animal practice: A retrospective study in a veterinary teaching hospital in Italy. J. Antimicrob. Chemoter. 2011, 66, 920–927. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Barbarossa, A.; Rambaldi, J.; Miraglia, V.; Giunti, M.; Diegoli, G.; Zaghini, A. Survey on antimicrobial prescribing patterns in small animal veterinary practice in Emilia Romagna, Italy. Vet. Rec. 2017, 181, 69. [Google Scholar] [CrossRef] [PubMed]
  56. Chirollo, C.; Nocera, F.P.; Piantedosi, D.; Fatone, G.; Della Valle, G.; De Martino, L.; Cortese, L. Data on before and after the Traceability System of Veterinary Antimicrobial Prescriptions in Small Animals at the University Veterinary Teaching Hospital of Naples. Animals 2021, 11, 913. [Google Scholar] [CrossRef] [PubMed]
  57. De Briyne, N.; Atkinson, J.; Pokludová, L.; Borriello, S.P. Antibiotics used most commonly to treat animals in Europe. Vet. Rec. 2014, 175, 325. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Joosten, P.; Ceccarelli, D.; Odent, E.; Sarrazin, S.; Graveland, H.; Van Gompel, L.; Battisti, A.; Caprioli, A.; Franco, A.; Wagenaar, J.A.; et al. Antimicrobial Usage and Resistance in Companion Animals: A Cross-Sectional Study in Three European Countries. Antibiotics 2020, 9, 87. [Google Scholar] [CrossRef] [Green Version]
  59. Mazzaferro, E.M. Update on Canine Parvoviral Enteritis. Vet. Clin. N. Am. Small Anim. Pract. 2020, 50, 1307–1325. [Google Scholar] [CrossRef] [PubMed]
  60. Chaitman, J.; Ziese, A.-L.; Pilla, R.; Minamoto, Y.; Blake, A.B.; Guard, B.C.; Isaiah, A.; Lidbury, J.A.; Steiner, J.M.; Unterer, S.; et al. Fecal Microbial and Metabolic Profiles in Dogs with Acute Diarrhea Receiving Either Fecal Microbiota Transplantation or Oral Metronidazole. Front. Vet. Sci. 2020, 7, 192. [Google Scholar] [CrossRef] [PubMed]
  61. Tauro, A.; Beltran, E.; Cherubini, G.B.; Coelho, A.T.; Wessmann, A.; Driver, C.J.; Rusbridge, C.J. Metronidazole-induced neurotoxicity in 26 dogs. Aust. Vet. J. 2018, 96, 495–501. [Google Scholar] [CrossRef] [Green Version]
  62. Kilian, E.; Suchodolski, J.S.; Hartmann, K.; Mueller, R.S.; Wess, G.; Unterer, S. Long-term effects of Canine parvovirus infection in dogs. PLoS ONE 2018, 13, e0192198. [Google Scholar] [CrossRef]
  63. WHO (World Health Organization). Antimicrobial Resistance: Global Report on Surveillance 2014. 2014. Available online: https://www.who.int/antimicrobial-resistance/publications/surveillancereport/en/ (accessed on 7 October 2020).
  64. WHO (World Health Organization). WHO Guidelines on Use of Medically Important Antimicrobials in Food-Producing Animals. 2017. Available online: https://www.who.int/foodsafety/areas_work/antimicrobial-resistance/cia_guidelines/en/ (accessed on 7 October 2020).
  65. WHO (World Health Organization). Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. 2017. Available online: https://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant-bacteria/en/ (accessed on 7 October 2020).
  66. Effah, C.Y.; Sun, T.; Liu, S.; Wu, Y. Klebsiella pneumoniae: An increasing threat to public health. Ann. Clin. Microbiol. Antimicrob. 2020, 19, 1. [Google Scholar] [CrossRef]
  67. Alikhani, M.Y.; Asl, H.M.; Khairkhah, M.; Farajnia, S.; Aslani, M.M. Phenotypic and genotypic characterization of Escherichia Coli O111 serotypes. Gastroenterol. Hepatol. Bed Bench 2011, 4, 147–152. [Google Scholar] [PubMed]
  68. Kim, S.; Kim, S.-H.; Kim, J.; Shin, J.-H.; Lee, B.-K.; Park, M.-S. Occurrence and distribution of various genetic structures of class 1 and class 2 integrons in Salmonella enterica isolates from foodborne disease patients in Korea for 16 years. Foodborne Pathog. Dis. 2011, 8, 319–324. [Google Scholar] [CrossRef] [PubMed]
  69. Gaastra, W.; Kusters, J.G.; van Duijkeren, E.; Lipman, L.J.A. Escherichia fergusonii. Vet. Microbiol. 2014, 172, 7–12. [Google Scholar] [CrossRef] [PubMed]
  70. Gullett, J.C.; Westblade, L.F.; Green, D.A.; Whittier, S.; Burd, E.M. The Brief Case: Too beta to be a “B”. J. Clin. Microbiol. 2017, 55, 1604–1607. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Pinho, M.D.; Matos, S.C.; Pomba, C.; Lübke-Becker, A.; Wieler, L.H.; Preziuso, S.; Melo-Cristino, J.; Ramirez, M. Multilocus sequence analysis of Streptococcus canis confirms the zoonotic origin of human infections and reveals genetic exchange with Streptococcus dysgalactiae subsp. equisimilis. J. Clin. Microbiol. 2013, 51, 1099–1109. [Google Scholar] [CrossRef] [Green Version]
  72. Sato, Y.; Kuwamoto, R. A case of canine salmonellosis due to Salmonella infantis. J. Vet. Med. Sci. 1999, 61, 71–72. [Google Scholar] [CrossRef] [Green Version]
  73. Chomel, B.B. Emerging and Re-Emerging Zoonoses of Dogs and Cats. Animals 2014, 4, 434–445. [Google Scholar] [CrossRef] [Green Version]
  74. Purpari, G.; Mira, F.; Di Bella, S.; Di Pietro, S.; Giudice, E.; Guercio, A. Investigation on Canine parvovirus circulation in dogs from Sicily (Italy) by biomolecular assay. Acta Vet. Beogr. 2018, 68, 80–94. [Google Scholar]
  75. Touihri, L.; Bouzid, I.; Daoud, R.; Desario, C.; El Goulli, A.F.; Decaro, N.; Ghorbel, A.; Buonavoglia, C.; Bahloul, C. Molecular characterization of canine parvovirus-2 variants circulating in Tunisia. Virus Genes 2009, 38, 249–258. [Google Scholar] [CrossRef]
  76. Battilani, M.; Modugno, F.; Mira, F.; Purpari, G.; Di Bella, S.; Guercio, A.; Balboni, A. Molecular epidemiology of Canine parvovirus type 2 in Italy from 1994 to 2017: Recurrence of the CPV-2b variant. BMC Vet. Res. 2019, 15, 393. [Google Scholar] [CrossRef]
  77. Ogbu, K.I.; Mira, F.; Purpari, G.; Nwosuh, C.; Loria, G.R.; Schirò, G.; Chiaramonte, G.; Tion, M.T.; Di Bella, S.; Ventriglia, G.; et al. Nearly full-length genome characterization of Canine parvovirus strains circulating in Nigeria. Transbound. Emerg. Dis. 2020, 67, 635–647. [Google Scholar] [CrossRef] [Green Version]
  78. Pérez, R.; Calleros, L.; Marandino, A.; Sarute, N.; Iraola, G.; Grecco, S.; Blanc, H.; Vignuzzi, M.; Isakov, O.; Shomron, N.; et al. Phylogenetic and genome-wide deep-sequencing analyses of Canine parvovirus reveal co-infection with field variants and emergence of a recent recombinant strain. PLoS ONE 2014, 9, e111779. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Martella, V.; Decaro, N.; Elia, G.; Buonavoglia, C. Surveillance activity for Canine parvovirus in Italy. J. Vet. Med. B Infect. Dis. Vet. Public Health 2005, 52, 312–315. [Google Scholar] [CrossRef] [PubMed]
  80. Barrett, T.; Visser, I.K.; Mamaev, L.; Goatley, L.; van Bressem, M.F.; Osterhaust, A.D. Dolphin and porpoise morbilliviruses are genetically distinct from phocine distemper virus. Virology 1993, 193, 1010–1012. [Google Scholar] [CrossRef] [PubMed]
  81. Hu, R.L.; Huang, G.; Qiu, W.; Zhong, Z.H.; Xia, X.Z.; Yin, Z. Detection and differentiation of CAV-1 and CAV-2 by polymerase chain reaction. Vet. Res. Commun. 2001, 25, 77–84. [Google Scholar] [CrossRef] [PubMed]
  82. Pratelli, A.; Martella, V.; Decaro, N.; Tinelli, A.; Camero, M.; Cirone, F.; Elia, G.; Cavalli, A.; Corrente, M.; Greco, G.; et al. Genetic diversity of a canine coronavirus detected in pups with diarrhoea in Italy. J. Virol. Methods 2003, 110, 9–17. [Google Scholar] [CrossRef]
  83. Freeman, M.M.; Kerin, T.; Hull, J.; McCaustland, K.; Gentsch, J. Enhancement of detection and quantification of rotavirus in stool using a modified real-time RT-PCR assay. J. Med. Virol. 2008, 80, 1489–1496. [Google Scholar] [CrossRef]
  84. CLSI. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, 4th ed.; CLSI Supplement VET08; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018; ISBN 978-1-68440-010-2. [Google Scholar]
  85. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 25th ed.; CLSI supplement M100-S25; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2015; ISBN 978-1-56238-989-5/978-1-56238-990-1. [Google Scholar]
  86. Kim, J.; Jeon, S.; Rhie, H.; Lee, B.; Park, M.; Lee, H.; Lee, J.; Kim, S. Rapid Detection of Extended Spectrum β-Lactamase (ESBL) for Enterobacteriaceae by use of a Multiplex PCR-based Method. Infect. Chemother. 2009, 41, 181–184. [Google Scholar] [CrossRef] [Green Version]
  87. Vitale, M.; Gaglio, S.; Galluzzo, P.; Cascone, G.; Piraino, C.; Di Marco Lo Presti, V.; Alduina, R. Antibiotic Resistance Profiling, Analysis of Virulence Aspects and Molecular Genotyping of Staphylococcus aureus Isolated in Sicily, Italy. Foodborne Pathog. Dis. 2018, 15, 177–185. [Google Scholar] [CrossRef] [Green Version]
  88. Baums, C.G.; Schotte, U.; Amtsberg, G.; Goethe, R. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet. Microbiol. 2004, 100, 11–16. [Google Scholar] [CrossRef]
  89. Bai, J.; Paddock, Z.D.; Shi, X.; Li, S.; An, B.; Nagaraja, T.G. Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Foodborne Pathog. Dis. 2012, 9, 541–548. [Google Scholar] [CrossRef] [PubMed]
Table
Table 1. Bacteria isolated from dogs with canine parvovirus infection.
Table 1. Bacteria isolated from dogs with canine parvovirus infection.
SpeciesFamilyOrderNumber of
Isolates		Tissue Sample
Gram-negative
Escherichia coliEnterobacteriaceaeEnterobacteriales19Brain, heart, intestine, kidney, lymph nodes, liver, spleen
Klebsiella pneumoniae4Brain, intestine, liver, spleen
Enterobacter cloacae2Brain, intestine
Enterobacter gergoviae2Brain, intestine
Escherichia fergusonii1Intestine
Proteus mirabilis1Intestine
Salmonella enterica1Intestine
Pseudomonas
aeruginosa		PseudomonadaceaePseudomonadales1Intestine
Gram-positive
Clostridium perfringensClostridiaceaeClostridiales3Intestine, liver, spleen
Enterococcus faeciumEnterococcaceaeLactobacillales2Brain, lymph nodes
Enterococcus faecalis1Intestine
Streptococcus canisStreptococcaceaeLactobacillales2Brain, lung
Streptococcus
pseudoporcinus		1Intestine
Staphylococcus lentusStaphylococcaceaeBacillales1Lung
Staphylococcus sciuri1Brain
Staphylococcus xylosus1Brain
Table
Table 2. Antibiotic sensitivity results derived with the Kirby-Bauer method for the Gram-negative strains (n = 31).
Table 2. Antibiotic sensitivity results derived with the Kirby-Bauer method for the Gram-negative strains (n = 31).
Bacterial IsolatesDog ID.AMCAMPCFRCLCXMCVNCROCEQMETATMIPMSPDACNVACTENRMARMPZCDOSXT
Escherichia coli
(n = 19)		1IRRRRRIRRSSR aR aRR aSSSRSIR
3IRRRRRSRRSIR aR aRR aISSRSSS
4IRRRRRRRRRIR aR aIR aIISRSIR
5SSRRRISRRISR aR aIR aSSSRSSS
6SSRRRISRRISR aR aRR aSSSRSSS
7SRRRRISRRSSR aR aRR aSSSRSRR
8SRRRRISRRISR aRaRR aSSSRSSR
9IRRRRRSRRISR aR aIR aSSSRSSS
10RRRRRRSRRSSR aR aRR aSRRRRRR
11IRRRRISRRISR aR aRR aSRRRSSS
13SIRRRRSRRRSR aR aIR aIISRSRS
14SRSRRSSRRSSR aR aRR aSSSRIRR
15SSSRRSSRRSIR aR aSR aSSSRSSS
16SSRSRRSRRRIR aR aRR aSSSRSSS
17SSSSRSSRRISR aR aIR aSSSRSSS
19RRRRRRRRRRSR aR aRR aSRRRRIR
20SSSSRSSRRSSR aR aSR aSSSRSSS
22RRRRRISRRRRR aR aRR aSRRRRIS
23SSSSRSSRRRSR aR aSR aSISRSSS
Klebsiella pneumoniae
(n = 4)		6SR aRRRSSRRSSR aR aRR aSSSRSIR
10IR aRRRRSRRSSR aR aSR aSISRSRS
12IR aRRRRRRRRSR aR aSR aSISRSRR
22RR aRRRRRRRRSR aR aRR aSRIRSIR
Enterobacter cloacae
(n = 2)		2RR aRRRRRRRRSR aR aSR aSIRRSIR
13SS aRRRISRRISR aR aIR aSSIRSRS
Enterobacter gergoviae
(n = 2)		5SR aRRRISRRISR aR aSR aSISRSIS
9SR aRSRSSRRSSR aR aSR aSSSRSSS
Escherichia fergusoni
(n = 1)		21SSSSRSSRRSSR aR aSR aRSSRSRS
Proteus mirabilis
(n = 1)		20SSSSRSSRRSSR aR aSR aSSSRSR aS
Salmonella enterica
(n = 1)		3SRRRRISRRISR aR aRR aISSRSSS
Pseudomonas aeruginosa
(n = 1)		12R aR aR aR aR aRSRRRIR aR aSR aSSSRS aS aR
Amoxicillin-clavulanic acid (AMC); ampicillin (AMP); cefadroxil (CFR); cephalexin (CL); cefuroxime (CXM); cefovecin (CVN); ceftriaxone (CRO); cefquinome (CEQ); methicillin (MET); aztreonam (ATM); imipenem (IPM); spiramycin (SP); clindamycin (DA); gentamicin (CN); vancomycin (VA); colistin sulfate (CT); enrofloxacin (ENR); marbofloxacin (MAR); metronidazole (MPZ); chloramphenicol (C); doxycycline (DO); sulfamethoxazole + trimethoprim (SXT). S: sensible; R: resistant; I: intermediate; a Intrinsic resistance.
Table
Table 3. Antibiotic sensitivity results derived with Kirby–Bauer method for the Gram-positive strains (n = 12).
Table 3. Antibiotic sensitivity results derived with Kirby–Bauer method for the Gram-positive strains (n = 12).
Bacterial IsolatesDog ID.AMCAMPCFRCLCXMCVNCROCEQMETATMIPMSPDACNVACTENRMARMPZCDOSXT
Clostridium perfringens
(n = 3)		2SRRRRRRRRR aSRRRSR aISRSIR
3SSSSRISRSR aSISRSR aSSSSSR
20SSSSSRSRSR aSISRSR aSSSSSS
Enterococcus faecium
(n = 2)		4IRR aR aR aR aR aR aRR aRI aRRSR aRRRSII a
12IRR aR aR aR aR aR aRR aRI aIRSR aRRRSIR a
Enterococcus faecalis
(n = 1)		9SSS aR aR aR aIaR aRR aSR aR aSSR aRRRSRR a
Staphylococcus lentus
(n = 1)		20SSSSSSSRS aR aSIISSR aSSRSSS
Staphylococcus sciuri
(n = 1)		10SSSSRSSRS aR aSIISSR aSSRSSS
Staphylococcus xylosus
(n = 1)		4SRRRRRIRR aR aSRRSSR aRSRSSS
Streptococcus canis
(n = 2)		1SIISSSSRSR aSIRRSR aISRSSS
5SSSSRRSRSR aSISISR aISRSRS
Streptococcus pseudoporcinus
(n = 1)		18SSSSRISRSR aSIIISR aIIRSSS
Amoxicillin-clavulanic acid (AMC); ampicillin (AMP); cefadroxil (CFR); cephalexin (CL); cefuroxime (CXM); cefovecin (CVN); ceftriaxone (CRO); cefquinome (CEQ); methicillin (MET); aztreonam (ATM); imipenem (IPM); spiramycin (SP); clindamycin (DA); gentamicin (CN); vancomycin (VA); colistin sulfate (CT); enrofloxacin (ENR); marbofloxacin (MAR); metronidazole (MPZ); chloramphenicol (C); doxycycline (DO); sulfamethoxazole + trimethoprim (SXT). S: sensible; R: resistant; I: intermediate; a Intrinsic resistance.
Table
Table 4. β-lactamase genes, and antibiotic sensitivity results derived with the Kirby–Bauer method for the Enterobacteriaceae isolates.
Table 4. β-lactamase genes, and antibiotic sensitivity results derived with the Kirby–Bauer method for the Enterobacteriaceae isolates.
Dog ID.Bacterial IsolatesSourceβ-Lactamase GenesAMCAMPCFRCLCXMCVNCROCEQMETATMIPM
1E. coliIntestineblaTEM, blaCMY-II, blaCTX-M-IIRRRRRIRRSS
2E. cloacaeIntestineblaTEM, blaDHARR aRRRRRRRRS
3S. entericaIntestinenegativeSRRRRISRRIS
E. coliIntestinenegativeIRRRRRSRRSI
4E. coliIntestineblaTEM, blaCTX-M-IIIRRRRRRRRRI
5E. gergoviaeBrainnegativeSRaRRRISRRIS
E. coliIntestinenegativeSSRRRISRRIS
6E. coliIntestinenegativeSSRRRISRRIS
K. pneumoniaeBrainblaTEM, blaSHV, blaCTX-M-IISR aRRRSSRRSS
7E. coliIntestinenegativeSRRRRISRRSS
8E. coliIntestineblaTEMSRRRRISRRIS
9E. coli (0111, eae)IntestineblaTEM, blaCTX-M-IIIRRRRRSRRIS
E. gergoviaeIntestineblaSHVSR aRSRSSRRSS
10E. coliIntestinenegativeRRRRRRSRRSS
K. pneumoniaeBrainblaSHVIR aRRRRSRRSS
11E. coliIntestineblaTEM, blaOXAIRRRRISRRIS
12K. pneumoniaeIntestineblaTEM, blaOXA, blaSHVblaCTX-M-IIIR aRRRRRRRRS
13E. coliIntestinenegativeSIRRRRSRRRS
E. cloacaeBrainnegativeSS aRRRISRRIS
14E. coliIntestineblaTEM, blaCTX-M-IISRSRRSSRRSS
15E. coliIntestinenegativeSSSRRSSRRSI
16E. coliIntestinenegativeSSRSRRSRRRI
17E. coliIntestinenegativeSSSSRSSRRIS
19E. coliIntestineblaOXA, blaCTX-M-IIRRRRRRRRRRS
20E. coliIntestinenegativeSSSSRSSRRSS
P. mirabilisIntestinenegativeSSSSRSSRRSS
21E. fergusoniiIntestineblaCTX-M-IISSSSRSSRRSS
22E. coliIntestineblaTEM, blaOXA, blaSHV, blaCTX-M-IIRRRRRISRRRR
K. pneumoniaeIntestineblaTEM, blaOXA, blaSHV, blaCTX-M-IIRR aRRRRRRRRS
23E. coliIntestinenegativeSSSSRSSRRRS
Amoxicillin–clavulanic acid (AMC); ampicillin (AMP); cefadroxil (CFR); cephalexin (CL); cefuroxime (CXM); cefovecin (CVN); ceftriaxone (CRO); cefquinome (CEQ); methicillin (MET); aztreonam (ATM); imipenem (IPM); a Intrinsic resistance.
Table
Table 5. Multidrug-resistance evaluation of 27 strains of the Enterobacteriaceae family.
Table 5. Multidrug-resistance evaluation of 27 strains of the Enterobacteriaceae family.
Bacterial IsolatesDog IdPenicillinsCephalosporinsCarbapenemAminoglycosidesFluoroquinolonesChloramphenicolSulfonamidesBeta
Lactams		Tetracycline
AMCAMPCLCVNIPMCNENRMARCSXTMETPIPDOTE
KBMICKBMICKBMICKBMICKBMICKBMICKBMICKBMICKBMICKBMICKBMICKBMIC
E. coli (n = 16)1IRRRRRRRSSRSSSSSSSRSRRIR
3ISRSRRRSISRRSSSSSSSSRSSS
4IRRRRRRRISIRISSSSSRRRRIR
5SSSSRRISSSIRSSSSSSSSRSSS
6SSSSRRISSSRRSSSSSSSSRSSS
7SSRRRRISSSRSSSSSSSRRRRRR
8SSRRRRISSSRRSSSSSSRRRRSS
9ISRRRRRSSSIRSSSSSSSSRRSS
10RRRRRRRSSSRSRRRRRRRRRRRR
11IRRRRRISSSRRRRRRSSSSRRSS
13SSISRRRSSSIRISSSSSSSRRRS
14SSRRRRSSSSRSSSSSIIRRRSRR
15SSSSRRSSISSRSSSSSISSRRSS
16SSSSSRRSISRRSSSSSSSSRSSS
17SSSSSRSSSSIRSSSSSSSSRSSS
19RRRRRRRSSSRRRSRRRSRSRSIS
20RSRRRSISRSRSRRRRRRSSRSIR
Klebsiella pneumoniae (n = 4)6SSR aR aRSSSSSRRSSSSSSRRRRIR
10SIR aR aRSRSSSSSISSSSSSSRRRR
12IIR aR aRRRRSSSSIISSSSRRRRRR
20RIR aR aRRRRSSRRRRIISSRRRRIR
Enterobacter cloacae (n = 2)2RRR andRRRSSSSSIIRSSSRRRRIR
13SRS andRRISSSISSSISSSSSRSRR
Enterobacter gergoviae (n = 2)5SRR andRRISSSSSISSSSSSSRSIS
9SRR andSRSSSSSSSSSSSSSSRISS
Escherichia fergusoni (n = 1)23SSSSSSSSSSSSSSSSSSSSRSRS
Proteus mirabilis (n = 1)22SSSSSISSSSSSSSSSSSSSRSR aR
Salmonella enterica (n = 1)3SSRSRSISSSRSSSSSSSSSRSSS
Amoxicillin + clavulanic acid (AMC); ampicillin (AMP); cephalexin (CL); cefovecin (CVN); imipenem (IPM); gentamicin (CN); enrofloxacin (ENR); marbofloxacin (MAR); chloramphenicol (C); sulfamethoxazole + trimethoprim (SXT); methicillin (MET); piperacillin (PIP); doxycycline (DO); tetracycline (TE); a Intrinsic resistance; nd: not determined.
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Schirò, G.; Gambino, D.; Mira, F.; Vitale, M.; Guercio, A.; Purpari, G.; Antoci, F.; Licitra, F.; Chiaramonte, G.; La Giglia, M.; et al. Antimicrobial Resistance (AMR) of Bacteria Isolated from Dogs with Canine Parvovirus (CPV) Infection: The Need for a Rational Use of Antibiotics in Companion Animal Health. Antibiotics 2022, 11, 142. https://doi.org/10.3390/antibiotics11020142

AMA Style

Schirò G, Gambino D, Mira F, Vitale M, Guercio A, Purpari G, Antoci F, Licitra F, Chiaramonte G, La Giglia M, et al. Antimicrobial Resistance (AMR) of Bacteria Isolated from Dogs with Canine Parvovirus (CPV) Infection: The Need for a Rational Use of Antibiotics in Companion Animal Health. Antibiotics. 2022; 11(2):142. https://doi.org/10.3390/antibiotics11020142

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Schirò, Giorgia, Delia Gambino, Francesco Mira, Maria Vitale, Annalisa Guercio, Giuseppa Purpari, Francesco Antoci, Francesca Licitra, Gabriele Chiaramonte, Maria La Giglia, and et al. 2022. "Antimicrobial Resistance (AMR) of Bacteria Isolated from Dogs with Canine Parvovirus (CPV) Infection: The Need for a Rational Use of Antibiotics in Companion Animal Health" Antibiotics 11, no. 2: 142. https://doi.org/10.3390/antibiotics11020142

APA Style

Schirò, G., Gambino, D., Mira, F., Vitale, M., Guercio, A., Purpari, G., Antoci, F., Licitra, F., Chiaramonte, G., La Giglia, M., Randazzo, V., & Vicari, D. (2022). Antimicrobial Resistance (AMR) of Bacteria Isolated from Dogs with Canine Parvovirus (CPV) Infection: The Need for a Rational Use of Antibiotics in Companion Animal Health. Antibiotics, 11(2), 142. https://doi.org/10.3390/antibiotics11020142

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