COMPARATIVE RFLP-ITS ANALYSIS BETWEEN ENTEROBACTER CLOACAE STRAINS ISOLATED FROM PLANTS AND CLINICAL ORIGIN

ANÁLISE COMPARATIVA DE RFLP-ITS ENTRE LINHAGENS DE ENTEROBACTER CLOACAE ISOLADAS DE PLANTAS E DE ORIGEM CLÍNICO-HOSPITALAR

ABSTRACT

Nineteen strains of Enterobacter cloacae including ten isolates from vegetables and eight from clinical origin and one from spring water were analysed by 16S-23S intergenic transcribed spacer (ITS) primers. Type strains of E. cloacae (ATCC 13047), E. dissolvens (ICMP 1570), and E. nimipressuralis (ICMP 1577) were included for comparative purposes. Also, the strains were tested for pathogenicity on onion bulbs. PCR-RFLP results showed that E. cloacae strains isolated from plants were homogeneous presenting close similarity among them, whereas the strains from clinical samples were heterogeneous. Pathogenicity tests revealed that the group comprising bacteria from clinical origin also showed capability of were able to induce a collapse of the inner scales of onion bulbs.

KEY WORDS:
Pathogenicity; PCR; RFLP analysis.

RESUMO

Dezenove linhagens de Enterobacter cloacae incluindo dez isolados de vegetais, oito isolados clínicos e um de água de fonte foram analisados por meio de PCR-RFLP utilizando primers intergênicos das regiões espaçadoras 16S-23S. Linhagens tipo de E. cloacae (ATCC 13047), E. dissolvens (ICMP 1570) e E. nimipressuralis (ICMP 1577) foram incluídas a título de comparação. A técnica de PCR-RFLP demonstrou que as linhagens vegetais aparecem homogêneas e são claramente distinguidas, enquanto que as de origem clínica mostraram heterogeneidade. Os testes de patogenicidade revelaram que o grupo das linhagens clínicas também induziu colapso interno nas escamas da cebola.

PALAVRAS-CHAVE:
Patogenicidade; PCR; análise por RFLP.

INTRODUCTION

Enterobacter cloacae is a bacterium widely distributed in nature, and has been recognized as a nosocomial pathogen, sometimes as a potential or sometimes even as a primary pathogen mainly due to itsability to develop resistance to antibiotics. Several reports of infectious in hospitals have been made, for instance in neonatal intensive care units, surgical wards, burn units, or caused by cross-infection and other types. Articles and revisions on this subject are widely available in the medical literature. Also, often the source of epidemics in hospital environment is associated with contaminated pharmaceutical products. In Brazil this fact was observed by PISANI et al. (1997).

In agriculture E. cloacae has been found in many insects such as symbiotic or entomopathogenic and in the surface of vegetables. Several reports have been made with E. cloacae strains in the biological control of phytopathogens, for instance Phytium spp., Sclerotinia sp., Rhizopus sp., Fusarium spp. and many others.

E. cloacae has emerged in recent years as a phytopathogen of important crops. A reliable evidence of the pathogenicity of E. cloacae for plants was noticed by COOTHER & DOWLING (1986) in stored onions. Earlier TANII et al. (1974) had isolated E. cloacae from rice seeds in association with Erwinia, Brevibacterium and Pseudomonas without confirming however involvement in the disease process. Further reports of E. cloacae phytopathogenicity were made by WICK eta l. (1987) concerning mung bean sprouts, by NISHIJIMA et al. (1987) who isolated the bacterium from papaya, by BISHOP & DAVIS (1990) and SCHWARTZ & OTTO (2000) who isolated it. NISHIJIMA et al. (1987) and BISHOP & DAVIS (1990) found in their studies that the type strain of E. cloacae from clinical origin were also pathogenic to papaya and onions, respectively. In Brazil, E. cloacae causing disease on plants was firstly observed by ROBBS et al. (1995) from samples of diseased melons collected at the northeast areas of the country, showing symptoms of pulp discoloration. Further isolations of the bacterium were made by ROBBS (C.F. Robbs, personal communication) from papaya, mangoes and lettuce.

The occurrence of bacterial species from the clinical samples environment on vegetables and vice-versa is a recognized fact. Besides E. cloacae, other species have been detected causing disease on plants or animals, for instance Pantoea agglomerans (MERGAERT et al.,1983; LINDH & URSING, 1986; GAVINI et al.,1989), Pseudomonas aeruginosa (LEBEDA et al., 1984), Agrobacterium sp. (FRENEY et al., 1985), Herbaspirillum “species 3” (BALDANI et al., 1996), Serratia marcescens (BERIAM et al., 1993; ROBBS et al., 1998; DORSEY et al., 2000), Burkholderia cepacia which is an important phytopathogen (BRADBURY, 1986) and frequently isolated from clinical (VAN LAER et al., 1998) as well as Burkholderia gladioli (HOARE & CANT, 1996).

A great number of typing schemes have been used for E. cloacae, including biotyping, serotyping, bacteriocin, protein profiles and phage typing. These methods however require an intensive work and lack resolution to detect small changes in the bacterial genome. Analyses based on DNA fingerprints are largely available and they have been employed with advantages in labor, time and precision. The arbitrarily amplified polymorphic DNA (AP-PCR) with randomic primers is able to detect slight genetic differences and has been used in various bacterial species for strains differentiation and epidemiological analysis. Molecular approaches such as the restriction fragment length polymorphism of the 16S-23S rDNA intergenic spacer region (RFLP-ITS) has been widely used to characterize bacterial strains at different taxa level. The ITS region is not under such high selective pressure as the 16S rDNA, another commonly used gene for phylogenetic purposes, and depending on the variation detected it might be a useful tool for discrimination at infraspecific level.

Although the genetic analysis of E. cloacae has been carried out by several authors, no studies were performed to investigate the relationship among E. cloacae strains coming from clinical and its comparison with isolates from vegetables, which could be useful in studies of nosocomial epidemics. Therefore, an experiment was carried out in order to investigate this relationship by using the RFLP-ITS, a more conserved feature. In addition, the phytopathogenic possibility of E. cloacae strains coming from clinical samples was also investigated.

MATERIAL AND METHODS

Bacterial strains and growth conditions - twenty IBSBF Culture Collection deposited strains of E. cloacae comprising then strains isolated from plants, eight strains from clinical cases, one strain from water source and the type strain of E. cloacae (ATCC 13047), are listed in Table 1. In addition, the type strains of Enterobacter dissolvens (ICMP 1570) and Enterobacter nimipressuralis (ICMP 1577) were included in this study since they were considered closely related to the type strain of E. cloacae (BRENNER et al., 1986; LINDH & URSING, 1991). All strains were grown on nutrient agar (NA) and incubated for 24-48h at 28^o C.

DNA extraction - genomic DNA was prepared from a loopful of cells grown for 24 h and washed once in phosphate buffer. The cell pellet was resuspended in 10 ml of TE buffer (40 mM Tris-HCl pH 8.0; 1 mM EDTA) and DNA was extracted according to PITCHER et al. (1989). DNA was resuspended in TE buffer and stored at -20^o C.

RFLP of the 16S-23S rDNA intergenic transcribed spacer - the spacer region between the 16S and 23S rDNA was amplified by using known primers for conserved regions: the 16S uni 330 and 23S uni 322 anti described by HONEYCUTT et al. (1995). The reaction was carried out in a final volume of 25 µL and contained 30-50 ng of DNA, 3 mM MgCl₂, 100 mM dNTP, 0.5 µM of each primer and 1.5 U of Taq polymerase (Cenbiot, Brazil). The cycling conditions were as described in HONEYCUTT et al. (1995). The amplification products were digested with Sau3 AI, HinfI or DdeI restriction enzymes and separated by electrophoresis in 7% polyacrilamide gel and visualized by silver staining (MORENO et al., 1985).

Data analysis - the banding pattern generated by RFLP-ITS was converted in binary matrices assigning 1 for presence and 0 for absence of a designed band. Similarity matrices were constructed by using (SJ/UPGMA). A dendrogram was constructed with the UPGMA (unweighted pair group method with average) with the program NTSYS-PC (ROHLF, 1989).

Pathogenicity test - pathogenicity tests were performed on mature onion bulbs to verify if strains isolated from clinical and from vegetables could induce disease symptoms. Five bulbs were inoculated through a syringe and hypodermic needle with about 0.5 mL of a bacterial suspension in distilled water of each strain with approximately 10⁸ colony forming unity per milliliter (CFU/mL) injected in tissues. Controls were inoculated with water. The inoculated onion bulbs were maintained at 36^o C in a humid chamber. Pathogenicity symptoms were recorded after a week according to the following scale: 0 = tissues remain undamaged and without discoloration; 1 = slight discolored tissues 1 cm around the inoculation point and tissues undamaged; 2 = inner scales discolored or flaccid; 3 = inner scales flaccid or discolored from top to the basis; 4 = flaccid and discolored tissues of half or entire bulb.

Table 1
Code and origin of Enterobacter cloacae strains used in this study.

Code IBSBF	Origin/host	Locality/source	Also held as
1140	Cucumis melo	Açu, RN	CTAA-35-L4*
1141	“ “	“ “	CTAA -35-L1
1142	“ “	“ “	CTAA -35-L2
1143	“ “	“ “	CTAA -35-L3
1189	“ “	“ “	CTAA -53-1
1186	Carica papaya	Linhares, ES	CTAA -51-1
1188	“ “	“ “	CTAA -51-2
1184	Mangifera indicae	Rio de Janeiro, RJ	CTAA -50-1
1185	“ “	“ “	CTAA -50-2
1291	Lactuca sativa	“ “	CTAA -3430
1187	Water source	Uberlândia, MG	CTAA -49-1
1176	Clinical	(CAISM) Campinas, SP
1177	“	“ “
1178	“	“ “
1231	“	Vitória, ES
1233	“	“ “
1249	“	IAL 24
1250	“	IAL 131
1251	“	IAL 133
	“	CCT 0456	ATCC 13047

ATCC= American Type Culture Collection, Rockville, Md., USA; CAISM= Unicamp, Campinas, Brazil; CCT= Coleção de Culturas Tropical, Campinas, Brazil; IAL= Instituto Adolfo Lutz, São Paulo, Brazil; IBSBF= Culture Collection of Phytobacteriology Section, Instituto Biológico, Campinas, Brazil.
• ^*
  Cultures CTAA were provided by Dr. C.F. Robbs.

Fig. 1
RFLP pattern of the 16S-23S ITS rDNA of Enterobacter cloacae and type strains of E. dissolvens and E. nimipressuralis after digestion with Dde I (A) and Sau 3 AI (B). M₂ 100 bp molecular weight marker.

Fig. 2
RFLP patterns of the 16S-23S ITS region of Enterobacter cloacae after digestion with Sau 3A I. M₂, 100 bp DNA Molecular Weight Marker.

RESULTS AND DISCUSSION

The amplification of 16S-23S ITS yielded a unique fragment of 1,000 base pairs approximately. Digestion of the PCR products with the restriction enzymes DdeI, HinfI and Sau3 AI revealed similarity among the majority of E. cloacae strains isolated from plants except for 1186 and 1291 strains. Slight differences were observed in the patterns obtained with DdeI or Sau3 AI. The digestion with HinfI generated most of the discriminative bands which could allow the clear differentiation between clinical and phytopathogenic strains (Fig. 1). The Sau3 AI digestions yielded a very uniform pattern for all E. cloacae strains and only E. cloacae ATCC 13047 showed one differential band (Fig. 2).

The data obtained from PCR-RFLP for the 16S-23S ITS region were combined for cluster analysis. Using UPGMA cluster analysis, the similarities among strains from plants ranging up to 95% and different groups were identified as shown in the dendrogram (Fig. 3). The clinical strains showed about 80% similarity to each other except for strain 1251, which is more related to plant strains. One interesting point is the fact that the type strain ATCC 13047 remained as a single branch with lower than 50% similarity to other E. cloacae strains investigated. Such a weak relationship of the type strain to other E. cloacae strains was also described by LINDH & URSING (1986). The type strains of E. dissolvens and E. nimipressuralis were closer to each other with approximately 90% similarity but with values lower than 50% in comparison with the type strain of E. cloacae ATCC 13047. Although the remarkable differences found in the relatively few strains of E. cloacae studied herein, it is evident that E. dissolvens and E. nimipressuralis showed a pattern that diverged significantly and provided evidence that these species are not related to E. cloacae. The strain 1291 was situated in a single branch with approximately 45% similarity with the other E. cloacae strains, suggesting that it could belong to another Enterobacter species.

The genetic diversity detected in E. cloacae is not a novelty. It has been described by several authors in epidemiological studies of nosocomial outbreaks, for instance GARAIZAR et al., (1991), HAERTL & BANDLOW (1993), HARTSTEIN et al. (1995) and DARINI et al. (1999), and a single patient may be infected with different strains (POILANE et al, 1993).

The E. cloacae strains examined from clinical origin clearly induced variable levels of symptoms on onion bulbs as did the strains isolated from vegetables, except for strain 1231, which showed no tissue alteration. Reisolations made from scales showing tissues discoloration symptoms yielded bacteria similar to those inoculated and the strains could be divided into four groups according to their capability of destroying the onion tissues, causing a brown discoloration and a decay of the inner scales. This feature is illustrated in Figure 4.

The first group (level 4) comprised strains 1140, 1143 and 1185; the second group (level 3) comprised strains 1184, 1187, 1188 and 1250; level 2 were 1142, 1176, 1233, 1291 and ATCC 13047; level 1 comprised strains 1141, 1177, 1178, 1186, 1249 and 1251. The type strains of E. dissolvens and E. nimipressuralis showed a similar degree of pathogenicity in the level 2.

Considering the clinical strains, the pathogenicity evaluation showed that most of the strains were classified in the intermediate levels except for strain 1250 which presented a particular strong pathogenicity reaction. The results indicated that clinical strains were able to induce symptoms in the plant species tested.

Fig. 3
Dendrogram (s_j/ UPGMA) derived from RFLP of the 16S-23S ITS region digested with Dde I, Hinf I and Sau 3A I restriction enzymes of Enterobacter cloacae and type strains of E. dissolvens and E. nimipressuralis. The letter before the number of the strains designates the origin: C (clinical), P (plants) and W (water).

Fig. 4
Pathogenicity levels of Enterobacter cloacae on onion bulbs after artificial inoculation. A, strain 1251 (level 1); B, strain 1184 (level2); C, strain 1250 (level 3); D, strain 1140 (level 4).

When bacterial strains isolated from plants were evaluated for pathogenicity interesting differences were observed and no correlation was found in the host origin and pathogenicity to onion. Also, the majority of these strains showed a marked phytopathogenicity at levels 3 and 4. The mode of infection of E. cloacae strains to onion bulbs remain undetermined, however it seems possible that enzymes production by the bacterial cells may affect the cell wall of the plant host by increasing the permeability of cell membranes as it occurs , for instance, in the soft-rot Erwinia (BARRAS et al., 1994).

Several epidemiological studies revealed that E. cloacae of an opportunistic pathogen and the reservoir for strains involved in nosocomial infections is the gastrointestinal tract of patients as well as contaminated water or parenteral nutrition and crosscontamination. The observations presented in this paper indicated that clinical strains of E. cloacae although differentiable from the plant strains by RFLPITS, were able to induce some kind of "symptom" and to multiply on onion tissue.

Indeed, E. cloacae has been isolated from postharvest vegetables (BARTOLONI et al., 1989), from vegetables and cooked meat samples in restaurants (SORIANO et al., 2001), and from manufactured yoghurts (CANGANELLA et al., 1999), and it is known that they persist in insects for at least three days (ARMSTRONG et al.,1989). These facts may contribute to the survival and dispersal of the bacterium by contaminating foods or materials and act as a "reservoir" in the hospital environment. In this manner, the phytopathogenicity capability of E.cloacae could be regarded as another presumable source of inoculum. Questions that arise are related to the host range (plants) of the pathogen, their survival on spoiled vegetables and in insects and the establishment of the minimal bacterium population required for a infection process. Also, the knowledge of the adhesive properties of the strains such as exopolyssacharide production and fimbriae (LIVRELLI et al., 1996) could provide more information useful aiming at the control of its populational expansion and prophylaxis measures.

Another point to be investigated is the patterns of antibiotic resistance in strains coming from food, plants and clinical samples which could provide (or not) a evidence that contaminated vegetables can indeed introduce the bacteria in the hospital environment.

ACKNOWLEDGMENT

We thank Dr. C.F. Robbs for supplying cultures.

REFERENCES

• ARMSTRONG, J.L.; PORTEOUS, L.A.; WOOD, N.D. The cutworm Peridroma saucia (Lepidoptera: Noctuidae) suports growth and transport of pBR322-bearing bacteria. Appl. Environ. Microbiol., Washington, v.55, p.22002205, 1989.
• BALDANI, J.I.; P OT, B.; KIRCHHOF, G.; F ALSEN, E.; B ALDANI, V.L.D.; OLIVARES, F.L.; HOSTE, B.; KERSTERS, K.; HARTMANN, A.; GILLIS, M.; DÖBEREINER, J. Emended descriptionnn of Herbaspirillim, inclusion of [Pseudomonas] rubrisubalbicans, a mild plant pathogen, as Herbaspirillum rubrisulbicans comb. Nov.; and classification of a group of clinical isolates (EF group1) as Herbaspirillum species 3. Int. J. Syst. Bacteriol., Washington, v.40, p.802-810, 1996.
• BARRAS, F.; VAN GIJSEGEM, F.; CHATTERJEE, A.K. Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu. Rev. Phytopathol., Palo Alto, v.32, p.201-234, 1994.
• BARTOLONI, A.; AQUILINI, D.; PARADISI, F. Enterobacteriaceae recovered from vegetables in Florence. Igiene-Moderna, Roma, v.91, p.1164-1168, 1989.
• BERIAM, L.O.S.; SINIGAGLIA, C.; RODRIGUES NETO, J. Serratia marcescens associada à podridão de bulbos de cebola armazenada. Fitopatol. Bras., Fortaleza, v18, supl., p.296, 1993.
• BISHOP, A.L. & DAVIS, R.M. Internal decay of onions caused by Enterobacter cloacae. Plant Dis., St. Paul, v.74, p.692694, 1990.
• BRADBURY, J.F. Guide to plant pathogenic bacteria. Ferri Lane, Surrey: CAB Int. Micol. Institute, 1986. p.332.
• BRENNER, D.J.; MCWHORTER, A.C.; KAI, A.; STEIGERWALT, A.G.; FARMER III, J.J. Enterobacter asburiae sp. nov., a new species found in clinical specimens, and reassignment of Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterobacter nimipressuralis comb. nov. J. Clin. Microbiol., Washington, v.23, p.1114-1120, 1986.
• CANGANELLA, F.; NESPICA, M.L.; GIONTELA, D.; TROVATELLI, D. Survival of Enterobacter cloacae and Pseudomonas paucimobilis in yoghurts manufactured from cow’s milk and soymilk during storage at different temperatures. Microbiol. Res., Jena, v.154, p.15-21, 1999.
• COOTHER, E. J. & D OWING, V. Bacteria associated with internal breakdown of onion and their possible role in disease expression. Plant Dis., St. Paul, v.35, p.329-336, 1986.
• DARINI, A.L.C.; MAGALHÃES, V.D.; LEVY, C.L.; BARTH, A.L.; COSCINA, A.L. Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae. Braz. J. Med. Biol. Res, Ribeirão Preto, v.32, p.1077-1081, 1999.
• DORSEY, G.; BORNEO, H.T.; SUN, S.J.; WELLS, J.; STEELE, L.; HOWLAND, K.; P EDREAE-REMINGTON, F.; BANGSBERG, D.R. A heterogeneous outbreak of Enterobacter cloacae and Serratia marcescens infections in a surgical intensive care unit. Infect. Control Hosp. Epidemiol., Thorofare, v.21, p.465-469, 2000.
• FRENEY, J.; GRUER, L.D.; B ORNSTEIN, N.; KIREDJIAN, M.; GUIVOUT, I.; LETOUZEY, M.N.; COMBE, C.; FLEURETTE, J. Septicemia caused by Agrobacterium sp. J. Clin. Microbiol., Washington, v.22, p.683-685, 1985.
• GARAIZAR, J.; KAUFMANN, M.E.; PITT, T.L. Comparison of ribotyping with conventional methods for the type identification of Enterobacter cloacae. J. Clin. Microbiol., Washington, v.29, p.1303-1307, 1991.
• GAVINI, F.; MERGAERT, J.; BEJI, A.; MIELCAAREK, C.; IZARD, D.; KERSTERS, K.; DE LEY, J. Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoae dispersa sp. nov. Int. J. Syst. Bacteriol, Washington, v.30, p.337-345, 1989.
• HAERTL, R. & BANDLOW, G. Epidemiological fingerprint of Enterobacter cloacae by small-fragment restriction endonuclease analysis and pulsed-field gel electrophoresis of genomic fragments. J. Clin. Microbiol., Washington, v.31, p.128-133, 1993.
• HARSTSTEIN, A.I.; CHETCHOTISAKD, P.; PHELPS, C.L.; LEMONTE, A.M. Typing of sequential bacterial isolates by pulsedfield gel electrophoresis. Diag. Microbiol. Infect. Dis., New York, v.22, p.309-314, 1995.
• HOARE, S. & CANT, J. Chronic glaucomatous disease presenting as severe septicemia due to Burkholderia gladioli. Clin. Infect. Dis., Chicago, v.23, p.411, 1996.
• HONEYCUTT, R.J.; SOBRAL, B.W.S.; MCCLELLAND, M. tRNA intergenic spacers reveal polymorphisms diagnostic for Xanthomonas albilineans. Microbiology, Reading, v.141, p. 3229-3239, 1995.
• LEBEDA, A.; KÜDELA, V.; JEDLICKOVÁ, Z. Pathogenicity of Pseudomonas aeruginosa for plants and animals. Acta Phytopathol. Acad. Sc. Hungariae, Budapest, v.19, p.271284, 1984.
• LINDH, E. & URSING, J. Clinical strains of Enterobacter agglomerans (synonims: Erwinia herbicola, Erwinia milletiae) identified by DNA-DNA hybrization. Acta Pathol. Microbiol. Immunol. Scand., Copenhagen, v.94, p.205-213, 1986.
• LINDH, E. & URSING, J. Genomic groups and biochemical profiles of clinical isolates of Enterobacter cloacae. Acta Pathol. Microbiol. Immunol. Scand., Copenhagen, v.99, p.507-514, 1991.
• LIVRELLI, V.; DE CHAMPS, C.; DI MARTINO, P.; DARFEUILLEMICHAUD, A.; F ORESTIER, C.; J OLY, B. Adhesive properties and antibiotic resistance of Klebsiella, Enterobacter, and Serratia clinical isolates involved in nosocomial infections. J. Clin. Microbiol., Washington, v.34, p.19631969, 1996.
• MERGAERT, J.; GAVINI, F.; KERSTERS, K.; LECLERC, H.; DE LEY, J. Phenotipic and protein similarities between strains of Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae from clinical or plant origin. Curr. Microbiol., New York, v.8, p.327-331, 1983.
• MORENO, M.R.; SMITH, J.F.; SMITH, R.V. Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie blue-silver stain procedure. Ann. Biochem., Paris, v.151, p.466-470, 1985.
• NISHIJIMA, K.A.; COUEY, H.M.; ALVAREZ, A.M. Internal yellowing, a bacterial disease of papaya fruits caused by Enterobacter cloacae. Plant Dis., St. Paul, v.71, p.10291034, 1987.
• PITCHER, D.G.; SANDERS, N.A.; OWEN, R.J. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters Appl. Microbiol., Bedford, v.8, p.151-156, 1989.
• PISANI, B.; SIMÕES, M.; PRANDI, G.M.A.; ROCHA, M.M.M.; ABRAHÃO, C.E.C.; SALONI, I.; PIRES, M.B.L.; OLIVEIRA, M.R.C.; CEZAR, P.C. Surto de bacteremia pelo Enterobacter cloacae relacionado à administração de nutrição parenteral (NP), ocorrido na região de Campinas, São Paulo. In: CONGRESSO BRASILEIRO DE MICROBIOLOGIA, 19., 1997, Rio de Janeiro, RJ. Resumos Rio de Janeiro: 1997. p.81.
• POILANE, I.; CRUAD, P.; LACHASSINE, E.; GRIMONT, F.; GRIMONT, P.A.D.; COLLIN, M.; GAUDELUS, J.; TORTOLIN, J.C.; COLLIGNOS, A. Enterobacter cloacae cross-colonization in neonates demonstrated by ribotyping. Eur. J. Clin. Microbiol. Infect. Dis., Berlim, v.12, p820-826, 1993.
• ROBBS, C.F.; R ODRIGUES NETO, J.; BITTANCOURT, A.M.; ANDERSEN, P.M. Variabilidade em Serratia marcescens, um patógeno oportunista. Summa Phytopathol., Botucatu, v.24, p.80, 1998.
• ROBBS, C.F.; RODRIGUES NETO, J.; SILVA, O.F.; BERIAM, L.O.S. Deterioração de polpa de frutos de melão causada por Enterobacter cloacae. Fitopatol. Bras., Fortaleza, v.20, p.281, 1995.
• RHOLF, F.J. NTSYS-PC numerical taxonomy and multivariate analysis system version 150 New York: Exeter Publ., 1989.
• SCHWARTZ, H.F. & OTTO, K. First report of a bulb decay of onion by Enterobacter cloacae in Colorado. Plant Dis.,S t. Paul, v.84, p.808, 2000.
• SORIANO, J.M.; RICO, H.; MOLTÓ, J.C.; MAÑES, J. Incidence of microbial flora in lettuce, meat and Spanish potato omelette from restaurants. Food Microbiol., New York, v.18, p.159-163, 2001.
• TANII, A.; BABA, T.; HARIKI, T. Bacteria isolated from black rot rice grains. Ann. Phytopathol. Soc. Japan, Tokyo, v.40, p.309-318, 1974.
• VAN LAER, F.; R AES, D.; VANDAMME, P.; LAMMENS, C.; SION, J.P.; VRINTS, C.; SNOECK, J.; GOOSSENS, H. An outbreak of Burkholderia cepacia with septicemia on a cardiology ward. Infect. Control. Hosp. Epidemiol., Thorofare, v.19, p.112-113, 1998.
• WICK, R.L.; RANE, K.K.; SUTTON, D.K. Mung bean sprout disease caused by Enterobacter cloacae. Phytopathology, St. Paul, v.77, p.123, 1987.

Publication Dates

• Publication in this collection
  28 June 2024
• Date of issue
  Jul-Sep 2003

History

• Received
  13 Mar 2003
• Accepted
  17 Apr 2003