Pantoea agglomerans, a plant pathogen causing human disease

We present 53 pediatric cases of Pantoea agglomerans infections cultured from normally sterile sites in patients seen at a children's hospital over 6 years. Isolates included 23 from the bloodstream, 14 from abscesses, 10 from joints/bones, 4 from the urinary tract, and 1 each from the peritoneum and the thorax. P. agglomerans was most associated with penetrating trauma by vegetative material and catheter-related bacteremia.     

Phylogeny and Identification of Pantoea Species and Typing of Pantoea agglomerans Strains by Multilocus Gene Sequencing

Pantoea agglomerans and other Pantoea species cause infections in humans and are also pathogenic to plants, but the diversity of Pantoea strains and their possible association with hosts and disease remain poorly known, and identification of Pantoea species is difficult. We characterized 36 Pantoea strains, including 28 strains of diverse origins initially identified as P. agglomerans, by multilocus gene sequencing based on six protein-coding genes, by biochemical tests, and by antimicrobial susceptibility testing. Phylogenetic analysis and comparison with other species of Enterobacteriaceae revealed that the genus Pantoea is highly diverse. Most strains initially identified as P. agglomerans by use of API 20E strips belonged to a compact sequence cluster together with the type strain, but other strains belonged to diverse phylogenetic branches corresponding to other species of Pantoea or Enterobacteriaceae and to probable novel species. Biochemical characteristics such as fosfomycin resistance and utilization of d-tartrate could differentiate P. agglomerans from other Pantoea species. All 20 strains of P. agglomerans could be distinguished by multilocus sequence typing, revealing the very high discrimination power of this method for strain typing and population structure in this species, which is subdivided into two phylogenetic groups. PCR detection of the repA gene, associated with pathogenicity in plants, was positive in all clinical strains of P. agglomerans, suggesting that clinical and plant-associated strains do not form distinct populations. We provide a multilocus gene sequencing method that is a powerful tool for Pantoea species delineation and identification and for strain tracking.The genus Pantoea includes several species that are generally associated with plants, either as epiphytes or as pathogens, and some species can cause disease in humans. Pantoea agglomerans, the Pantoea species most commonly isolated from humans, is widely distributed in nature and has been isolated from numerous ecological niches, including plants, water, soil, humans, and animals. It is frequently associated with plants as an epiphyte or an endophyte, and some isolates have been reported to be tumorogenic pathogens (20, 51). As an opportunistic human pathogen, P. agglomerans can occur sporadically or in outbreaks. At the beginning of the 1970s, P. agglomerans (then called Enterobacter agglomerans) was implicated in a large U.S. and Canadian outbreak of septicemia caused by contaminated closures on bottles of infusion fluids; 25 hospitals were involved, with 378 cases (34). Since then, P. agglomerans bacteremia has also been described in association with the contamination of intravenous fluid, parenteral nutrition, the anesthetic agent propofol, blood products, and transference tubes used for intravenous hydration (2-4, 22, 36). P. agglomerans has been recovered from joint fluids of patients with arthritis, synovitis, or osteomyelitis (7). Infection often occurs after injuries with plant thorns, wood slivers, or wooden splinters (7, 8, 16, 30, 40, 49). Cases of peritonitis due to P. agglomerans have also been reported (15, 31). Finally, P. agglomerans, which is known to colonize cotton and cotton plants heavily, is associated with cotton fever, a benign febrile syndrome seen in intravenous drug abusers (14).Seven Pantoea species are currently distinguished: P. agglomerans, the type species of the genus; Pantoea ananatis; Pantoea stewartii (divided into the two subspecies Pantoea stewartii subsp. stewartii, the agent of Stewart''s vascular wilt in maize and sweet corn, and Pantoea stewartii subsp. indologenes); Pantoea dispersa; Pantoea citrea; Pantoea punctata; and Pantoea terrea (18, 20, 28, 37). The Pantoea agglomerans complex was previously designated Erwinia herbicola or Enterobacter agglomerans (18). The biochemical heterogeneity of P. agglomerans and related strains and species renders identification difficult, even if several biochemical or nutritional characteristics distinguish the “Japanese group” (20) of Pantoea species (P. citrea, P. punctata, and P. terrea). Currently, confident identification is not achieved routinely.Precise knowledge of the phylogenetic relationships and the degree of genetic distinctness among Pantoea species is a prerequisite for their correct identification. Phylogenetic relationships among Pantoea species were initially based on 16S rRNA analysis (24, 39, 53), which showed that P. agglomerans, P. ananatis, and P. stewartii were closely related. The same result was obtained based on the three protein-coding genes atpD, carA, and recA (53). However, only one or a few strains per species were analyzed, and the phylogenetic relationships of the three “Japanese” species with the other Pantoea species have, to our knowledge, never been described. Hence, it is not clear whether Pantoea species are clearly demarcated and if the genus Pantoea forms only one phylogenetic branch.Defining bacterial species remains a challenge, especially given the fact that homologous recombination or lateral gene transfer can disturb species boundaries. Currently, the approach to defining bacterial species uses both genomic and phenotypic characteristics. The pragmatic values of 70% DNA-DNA reassociation and a difference of <5°C in the melting temperature have been proposed as a cutoff for species definition (50) but are technically challenging to obtain. Multilocus sequence analysis (MLSA) provides an alternative way to define species and to explore sequence discontinuities among them (19). Phylogenetic analysis of concatenated multiple protein-coding genes sometimes allow one to clearly separate species into sequence clusters that can be used to define species, even if borders may be fuzzy for highly recombinogenic species (17, 23). Moreover, the MLSA approach can be used to estimate homologous recombination among species, which is important for determining the reliability of molecular identification based on one or a few genes and for estimating the impact of homologous recombination or lateral gene transfer on the speciation process. So far, no MLSA approach has been reported for Pantoea species.Strain typing and population genetics studies are necessary for epidemiological purposes and to identify strains with important phenotypes such as virulence to plants or humans (11, 47). For example, it is important to determine if P. agglomerans strains differ in their abilities to infect humans or to cause specific diseases in plants. Currently, P. agglomerans strains can be differentiated using fluorescent amplified fragment length polymorphism (5) or pulsed-field gel electrophoresis (51). However, these methods do not provide unambiguous definition of clones and clonal families, and the results are generally difficult to compare among laboratories. Currently, the amount of diversity within P. agglomerans and its population structure are unknown. A widely accepted method for studying strain relationships is multilocus sequence typing (MLST) (33). It consists of sequencing internal portions of several protein-coding genes. This method provides unambiguous and portable data and allows one to compare data worldwide, which is necessary in order to achieve a comprehensive overview of strain diversity and epidemiological distribution (32). In addition, this method is suitable for studying strain phylogeny and allows one to address evolutionary questions at the strain level within species (11). In contrast to MLSA, MLST relies on the comparison of allelic profiles of strains within species, whereas MLSA uses concatenation of gene sequences to define boundaries and phylogenetic relationships between species.There are few data concerning the susceptibility of P. agglomerans to antimicrobial agents. In 1986, Muytjens et al. reported in vitro susceptibility data for eight species of Enterobacter, including 27 strains of E. agglomerans (38). The strains exhibited very variable susceptibilities to β-lactams, aminoglycosides, and quinolones. Hieng et al. (25) described a case of septicemia due to an Erwinia herbicola strain that was resistant to ampicillin, carbenicillin, and cephalothin (cefalotin) and susceptible to other antibiotics usually active on gram-negative bacilli. Cruz et al. (7) reported similar results. No β-lactamase was found among the Pantoea species. In 2000, a clinical isolate of P. agglomerans recovered from a patient with septic arthritis was reported to be highly resistant to fosfomycin (8).The objectives of this study were (i) to define the phylogenetic relationships among P. agglomerans strains, or strains that may be misidentified as P. agglomerans, and other species of Enterobacteriaceae; (ii) to characterize Pantoea species or phylogenetic clusters biochemically and for their susceptibilities to antimicrobial agents; and (iii) to develop and evaluate MLST for strain discrimination and determination of the population structure of P. agglomerans.     

Isolation of Pantoea agglomerans in two cases of septic monoarthritis after plant thorn and wood sliver injuries

Arthritis after plant injury is often apparently aseptic. We report two cases due to Pantoea agglomerans. In one case, the bacterium was isolated only from the pediatric blood culture media, BACTEC Peds Plus, monitored in BACTEC 9240, and not from the other media inoculated with the joint fluid. This procedure could help improve the diagnosis of septic arthritis.     

Serotypes of Pseudomonas aeruginosa from human and animal sources

Forty human isolates and twenty-eight animal isolates of Pseudomonas aeruginosa from Prince Edward Island area (Atlantic Canada) were serotyped using a kit consisting of 4 polyvalent O-group antisera and 17 monovalent O-type (serovar) antisera, in accordance with the International Antigenic Typing Scheme. The results showed a difference between humans/dogs (50%/48% group I) and pigs (71% group IV). Whereas O-serovar 9 and 1 appeared to be most involved in human and canine infections, O-serovar 3 was the predominant type in porcine infections.     

Distribution of serotypes of Nocardia asteroides from animal, human, and environmental sources.

The antigenic types of 129 isolates of Nocardia asteroides from diverse clinical, environmental, and geographic origins were determined. The majority of the isolates studied were of bovine (56) or human (44) origin; 11 were derived from six species of animals other than cattle, and 10 were isolated from environmental sources; the source of 8 strains could not be determined. Testing culture filtrate antigens against four standard reference sera in a gel diffusion precipitin test established the antigenic type of 95.3% of the isolates. After excluding strains that weighted the data because of common infection, the distribution of serotypes was examined according to the origin of the isolate. Type I was the most frequently encountered serotype (31.9%); types III (15.0%) and IV (20.4%) were also observed frequently, as was the antigenic mixture III + IV (14.2%). There was an apparent difference in frequency of type III and IV antigens among isolates of bovine and human origin; type III made up 20.0% of the bovine isolates and 13.6% of the human isolates, whereas type IV constituted 10.0% of bovine and 27.3% of human isolates.     

Evaluation of three differential media for detection of Enterobacter agglomerans (Erwinia herbicola).

Dextrin-fuchsine-sulfite medium (DFS), Rimler-Shotts agar (RS), and a new lysine-ornithine-mannitol agar (LOM) were tested for detection of Enterobacter agglomerans. In human stools, LOM and DFS were most sensitive at coliform-to-E. agglomerans ratios of less than or equal to 10(2) and E. agglomerans inocula of greater than 10(2) per plate. Both LOM and DFS detected one strain in 254 stools, but RS proved to be inhibitory.     

Characteristics of Yersinia enterocolitica and related species isolated from human, animal, and environmental sources.

During a 4-year period, 4,448 human, animal, and environmental samples collected in New York State were tested for the presence of Yersinia enterocolitica or related species. A total of 339 isolates were identified as Yersinia and characterized according to source, species, biogroup, serogroup, and, in some instances, phage type. Four new biogroups of Y. intermedia were characterized. Of 149 human isolates, 120 (80.5%) were identified as Y. enterocolitica, and 29 were identified as either Y. intermedia (12.1%), Y. frederiksenii (5.4%), or Y. kristensenii (2.0%). Of the other 190 isolates, recovered from animals and the environment, 54 (28.4%) were Y. enterocolitica and 136 were either Y. intermedia (62.6%), Y. frederiksenii (4.7%), Y. kristensenii (3.7%), or an undescribed Yersinia species (0.5%). Two established human pathogenic strains of Y. enterocolitica were recovered: 59 isolates (37 from an outbreak) of "American strain" (serogroup O:8, biogroups Niléhn 2, Wauters 1, and Knapp and Thal 2, phage type Xz) and 11 isolates of "Canadian strain" (serogroup O:3, biogroups Niléhn 4, Wauters 4, and Knapp and Thal 1, phage types IXb). This was the first documented isolation of the Canadian strain in the United States. Isolates of other strains implicated in human disease (serogroups 0:4,33, 0:5, O:6,31, O:7,8, and O:8) were also recovered from both human and nonhuman sources.     

Variant cysteine-rich surface proteins of Giardia isolates from human and animal sources.

Cloned Giardia isolates obtained from a sheep, a calf, and a human possessed a major membrane protein that showed marked intraspecific variations in size as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following surface biotinylation and radioiodination. Metabolic labeling with [35S] cysteine and electrophoretic analysis also revealed for each cloned isolate a predominant protein that corresponded in size to the major surface protein demonstrated by surface labeling techniques. Immunoprecipitation studies with a polyclonal antiserum specifically directed against the 90-kDa major cysteine-rich protein purified from a subclone of the sheep isolate (O2-4A1) showed that the cysteine-rich protein and the major surface protein are identical. The surface location of the antigen was further corroborated by the reaction of fluorescence-labeled antibodies raised against the 90-kDa O2-4A1 cysteine-rich protein with the entire surface of live trophozoites of the homologous clone. The ability of the cloned Giardia isolates to undergo variations of their cysteine-rich surface protein (CRISP) was demonstrated by the spontaneous appearance of new CRISPs in clonally derived populations during prolonged in vitro culturing and in cultures of the O2-4A1 clone that had survived treatment with the cytotoxic anti-90-kDa CRISP antiserum specific for the surface antigen of this clone. The surviving progeny were devoid of the original CRISP, as judged by resistance to the immune serum. Subsequent cysteine metabolic labeling of the recloned surviving trophozoites demonstrated a large number of new variants, each expressing a single CRISP that varied significantly in molecular weight from those in the different cloned lines. These studies suggest that the presence of CRISPs and their variations are not restricted to Giardia isolates obtained from humans but are universal phenomena among the Giardia duodenalis types of organisms.     

Host cell modulation by human, animal and plant pathogens

Members of the alpha-proteobacteria display a broad range of interactions with higher eukaryotes. Some are pathogens of humans, such as Rickettsia and Bartonella that are associated with diseases like epidemic typhus, trench fever, cat scratch disease and bacillary angiomatosis. Others like the Brucella cause abortions in pregnant animals. Yet other species have evolved elaborate interactions with plants; in this group we find both plant symbionts and parasites. Despite radically different host preferences, extreme genome size variations and the absence of toxin genes, similarities in survival strategies and host cell interactions can be recognized among members of the alpha-proteobacteria. Here, we review some of these similarities, with a focus on strategies for modulation of the host target cell.     

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.     

Growth and endotoxin production of Yersinia enterocolitica and Enterobacter agglomerans in packed erythrocytes.

Since 1987, the Centers for Disease Control investigated six cases of transfusion-associated sepsis. All six patients developed septic shock after receiving units of packed erythrocytes (PRBCs) contaminated with Yersinia enterocolitica (five patients) and Enterobacter agglomerans (one patient); three of the blood recipients died. We studied the growth and endotoxin production of Y. enterocolitica and E. agglomerans in units of PRBCs stored at 4 degrees C for 60 days. When PRBCs were inoculated with 0.1 to 1.0 CFU of these organisms per ml, both Y. enterocolitica and E. agglomerans entered log-phase growth 2 to 3 weeks after inoculation; generation times were 15 and 22 h, respectively. Endotoxin was first detected at 3 weeks following inoculation, and the concentration paralleled the log phase of growth of the strains tested. These data show that prolonged storage of PRBCs at 4 degrees C provides conditions that allow these two organisms to grow and subsequently produce high concentrations of endotoxin.     

Characterization of Salmonella enterica serotype Typhimurium isolates from human, food, and animal sources in the Republic of Ireland

A potential epidemic clone of Salmonella enterica serotype Typhimurium DT104, and the possible emergence of S. enterica serotype Typhimurium DT104b, has been identified from the characterization of 67 S. enterica serotype Typhimurium strains from three sources, human gastroenteritis isolates, isolates from food samples, and veterinary isolates, by antimicrobial resistance profiling, phage typing, and pulsed-field gel electrophoresis. Resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline was found in 77.6% of these strains.     

Comparison of Salmonella enterica serovar Heidelberg isolates from human patients with those from animal and food sources

Seventy-eight Salmonella enterica serovar Heidelberg isolates from humans were tested for antimicrobial susceptibility, resistance genes, and plasmids and genotyped by pulsed-field gel electrophoresis (PFGE). Most (88%) contained plasmids, and 47% were resistant to antimicrobials. The overall results were compared to those of previous S. Heidelberg studies of food- and animal-related sources, and multiple similarities were observed.     

Natural antibiotic susceptibility of Enterobacter amnigenus, Enterobacter cancerogenus, Enterobacter gergoviae and Enterobacter sakazakii strains

Objective   To investigate the natural susceptibility to 69 antimicrobial agents of 107 Enterobacter strains comprising E. amnigenus ( n  = 18), E. cancerogenus ( n  = 26), E. gergoviae ( n  = 28) and E. sakazakii ( n  = 35).
Methods   Minimal inhibitory concentrations (MICs) were determined with a microdilution procedure in Isosensitest broth and cation-adjusted Mueller–Hinton broth.
Results   All the species were naturally sensitive or intermediate to tetracyclines, amino-glycosides, numerous β -lactams (acylureidopenicillins, ticarcillin, ampicillin/sulbactam, several cephalosporins, carbapenems, aztreonam), quinolones, antifolates, chloramphenicol and nitrofurantoin. Natural resistance was found to penicillin G, oxacillin, several macrolides, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Species-related differences in natural susceptibility were found to some β -lactams, azithromycin and fosfomycin. Whereas E. gergoviae was the most susceptible species to azithromycin, E. cancerogenus was most susceptible to fosfomycin and was the only species showing natural resistance to amoxicillin, amoxicillin/clavulanic acid, cefaclor, cefazoline, loracarbef and cefoxitin. There were only minor medium-dependent differences in susceptibility to most antibiotics.
Conclusions   The present study establishes a database concerning the natural susceptibility of recently established Enterobacter species to a wide range of antibiotics, which can be applied for the validation of routine susceptibility test results. β -Lactam susceptibility patterns indicate the expression of species-specific β -lactamases expressed at high or low levels in all the species except E. sakazakii .