INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

The potential of members of the genus Acinetobacter to cause infection has been known for decades (7-9, 11, 18). However, only after improvement of species classification within the genus as a result of DNA-DNA hybridization studies (1, 3, 19) was it possible to gain insight into the ecology and clinical significance of individual Acinetobacter species (20). Of these, Acinetobacter baumannii (DNA group 2) has been isolated predominantly from clinical specimens of human origin and is clearly the main species associated with outbreaks of nosocomial infections (21). However, the reliable identification of this species in bacteriological laboratories is hampered by the close pheno- and genotypic relatedness of A. baumannii to three other species within the genus (5), two of which (unnamed DNA groups 3 and 13TU) (19) are known to also cause hospital-acquired infections (21). Due to the successful use of lipopolysaccharides (LPS) as taxonomic markers for a variety of gram-negative bacteria, we have decided to generate O-antigen-specific monoclonal antibodies (MAbs) against the LPS of Acinetobacter strains, with the aim of developing an identification scheme for this group of bacteria based on the chemical and antigenic structure of the O-polysaccharide of their LPS.

In a previous study, the pheno- and genotypic similarities among A. baumannii strains isolated in the Czech Republic were analyzed (12). Based on the results, these isolates could be classified into four groups: two relatively homogeneous groups of predominantly multiresistant strains (termed groups A and B) comprising both sporadic and outbreak-associated isolates, a heterogeneous group of other multiresistant strains, and a group of mainly susceptible strains (12). The features of groups A and B were found to be highly similar to those of two outbreak-related A. baumannii clonal groups, clones I and II, which were identified among hospital isolates in Northwestern Europe (6). In this study, we analyzed the O-antigenic relationship among these Czech strains, in comparison to a number of clone I and II strains, by using O-antigen-specific MAbs. The aim of the study was to gain insight into the prevalence of putative Acinetobacter O-serotypes (i.e., the O-antigen diversity), within the Czech Republic in particular, but also within the general clinical environment.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacteria. The Acinetobacter strains investigated in this study are listed in Table 1 (n = 65). They consisted of a selection of clinical isolates from the Czech Republic (n = 52) and Northwestern Europe (The Netherlands, United Kingdom, Belgium, and Denmark [n = 13]). Most strains were originally isolated from burn wounds, sputum, or urine. Forty-two Czech strains were identified previously as A. baumannii by ribotyping and characterized by antibiotic susceptibility, biotype, and plasmid profile (12). These strains were selected for this study from a set of 77 A. baumannii isolates (12) to be as heterogeneous as possible in their properties, geographical origin, and time of isolation, thus excluding multiple isolates of the same strain from one locality. Ten previously uncharacterized strains were added to broaden the geographical heterogeneity of the strains from the Czech Republic. The 13 strains from Northwestern Europe (Table 1) were identified previously as A. baumannii by DNA-DNA hybridization (6). The geno- and phenotypic characteristics of two of these strains, RUH 875 and RUH 134, have been compared recently to those of clinical isolates from the Czech Republic (12). For the present study, the additional Northwestern European strains and the additionally selected Czech strains were characterized for their ribotype, biotype, and antibiotic susceptibility as described previously (12). The presence of an 8.7-kb-plasmid, termed pAN1, was determined with a digoxigenin-labeled probe prepared from pAN1 of A. baumannii NIPH 632 (12). All strains used in this study were preserved in glycerol stocks at 80°C.

Whole-cell lysates and proteinase K digestion. Preparation of whole-cell lysates and proteinase K treatment of these lysates were performed as described in another study (14). They were stored at 20°C and heated (100°C, 5 min) prior to use.

MAbs. The MAbs used in this study are shown in Table 2. Their generation and serological characterization have been described in detail in other studies (15-17; R. Pantophlet, J. A. Severin, A. Nemec, L. Brade, L. Dijkshoorn, and H. Brade, submitted for publication; R. Pantophlet, L. Brade, and H. Brade, submitted for publication). They included MAbs against strains from the clinically more important species such as A. baumannii, DNA group 3, and DNA group 13TU, as well as MAbs against other species within the genus. All antibodies were stored at 20°C when not in use.

Serological assays. Dot blotting and Western blotting were performed as described previously (15) with proteinase K-treated bacterial lysates as antigens. Acid hydrolysis of membrane-bound LPS and detection of its lipid A moiety with antibody were performed as described elsewhere (13) with 1% acetic acid and MAb A6 against bisphosphoryl lipid A (10).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

The properties and epidemiological data of the 65 strains are shown in Table 1. Examples of EcoRI ribotypes are shown in Fig. 1. According to their ribotypes, all 10 additonally selected Czech strains were identified as A. baumannii. These strains were all resistant to at least three of nine antibiotics tested. Based on their EcoRI ribotypes and biotypes, seven of these strains (ribotype I, biotype 6 or 11) could be allocated to group A, and one strain (ribotype II, biotype 2) could be allocated to group B. The remaining two strains (ribotype X, biotype 2) were placed in the third group of multiresistant strains. A plasmid with a size of approximately 8.7 kb, termed pAN1 (12), was present in all of the additionally selected strains, which were placed in group A, as well as in the strain allocated to group B. 

View larger version (48K): [in this window] [in a new window]   FIG. 1.   Examples of EcoRI ribotypes observed for A. baumannii strains isolated in the Czech Republic. Lanes: 1, molecular weight marker (phage  DNA digested with HindIII and StyI); 2, strain NIPH 7; 3, strain NIPH 10; 4, strain NIPH 24; 5, strain NIPH 60; 6, strain NIPH 615. Strains NIPH 7 and NIPH 24 are representative of isolates allocated to groups A and B, respectively.

Proteinase K-digested lysates of all strains were then tested by dot blotting with the O-antigen-specific MAbs listed in Table 2. Strains giving positive reactions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting to confirm the reactivity observed in the dot blot with the respective MAb. O-antigen-reactivity was observed to be nearly exclusively with MAbs generated against A. calcoacetius and A. baumannii strains (Table 1). Group A isolates reacted with MAbs S48-3-13 (n = 17) and S51-3 (n = 5), generated against the O-antigens of an A. baumannii strain and an A. calcoaceticus strain, respectively. It is interesting in this context that clone I strains also reacted with MAb S48-3-13. Moreover, in both group A and clone I strains, plasmid pAN1 was found. Together with the previously noted similarities between group A and clone I reference strain RUH 875 (12), this finding strongly supports the hypothesis that group A and clone I strains are clonally related. The five remaining group A strains that reacted with MAb S51-3 may represent a subclonal group, judging from the similarities in their ribotypes and biotypes and the presence of plasmid pAN1 with the other group A strains. Only one other strain (NIPH 10, in the third group of multiresistant Czech strains) also reacted with MAb S51-3. Interestingly, plasmid pAN1 was also found in this strain, and its ribotype was highly similar to the EcoRI ribotype observed for group A and clone I strains (only one band position difference; Fig. 1). Plasmid pAN1 was originally isolated from A. baumannii strain NIPH 632 (12). Although its function and encoded properties are not known, it has been found in all (geographically highly diverse) strains with the EcoRI ribotype specific to clone I strains from Northwestern Europe, but rarely in other A. baumannii strains. This plasmid may thus serve as a clonal marker; however, further studies are needed to assess its role, if any, in the epidemicity of A. baumannii strains. Group B strains reacted exclusively with MAb S53-32, whereas clone II strains reacted primarily with MAb S48-3-17. A clonal relationship between these two groups of strains therefore seems unlikely. However, it must be noted here that clone II strains were originally delineated based primarily on their amplified fragment length polymorphism profile (6); two different EcoRI ribotype patterns were observed among these strains (Table 1) which were similar to the EcoRI ribotypes observed for two other outbreak-related strains that could not be allocated unambiguously to either clone I or II (6). Thus, the possibility of variants or subclones within this particular clonal group cannot be excluded. MAb reactivity with the other Czech strains included in this study was sporadic: two strains reacted with MAb S53-32, two reacted with MAb S48-3-17, and one reacted with MAb S53-25. The latter antibody was generated against the O-antigen of a strain belonging to genomic species 16, and its reactivity with A. baumannii strains would appear to be unusual at first glance. However, we have shown recently (14) that certain O-antigenic determinants may occur in different genomic species. This seems to be true as well for the epitope recognized by MAb S53-25. The generation of more O-antigen-specific antibodies against structurally defined epitopes of the O-polysaccharide chains will help clarify which epitopes determine species specificity and which do not. The high degree of reactivity among the homogeneous groups of multiresistant strains indicates limited O-antigen variation and supports the view that such strains are of a common clonal origin.

By using a method to visualize any LPS via its lipid A moiety with antibody in a Western blot following acid hydrolysis of the membrane-bound antigen (13), it was possible to define the putative O-serotypes of some strains that had not reacted with any of the MAbs used in the present study. Six novel banding patterns (labeled 1 to 6) were observed among 10 of the 17 strains, which did not react with any of the O-antigen-specific MAbs used in this study (Table 1 and Fig. 2). The lack of observation of a pattern for all strains has been noted in other studies (15, 16) and is probably because these strains have a reduced level of O-antigen expression or produce LPS that lacks an O-antigen. This is also an example how such antibodies may help analytical biochemists select those LPS that are worth being analyzed structurally.

View larger version (124K): [in this window] [in a new window]   FIG. 2.   Representative Western blot of A. baumannii strains that did not react with any of the O-antigen-specific MAbs used in this study following acid hydrolysis (1 h in 1% acetic acid at 100°C) of membrane-bound LPS and immunostaining with lipid A-specific MAb A6. Bacteria are, from left to right, strain NIPH 33 (lane 1), strain NIPH 335 (lane 2), strain NIPH 4 (lane 3), strain NIPH 45 (lane 4), strain NIPH 70 (lane 5), and strain NIPH 201 (lane 6).

The findings presented above suggest that certain A. baumannii serotypes may be more prevalent than others in hospital settings. In the Czech Republic, these would appear to be primarily the serotypes defined by MAbs S48-3-13, S53-32, and S51-3. In Northwestern Europe, the serotypes defined by MAbs S48-3-13 and S48-3-17 would appear to be most prevalent. Recent screening studies (R. Pantophlet, J. A. Severin, A. Nemec, L. Brade, L. Dijkshoorn, and H. Brade, submitted for publication) have shown that the serotypes defined by the MAbs mentioned above are also present in other Eastern European countries, such as Hungary, Bulgaria, and Poland. In Hungary and Bulgaria, the S51-3 serotype was found, whereas in Poland, the serotypes defined by MAbs S48-3-17 and S53-32 were identified among a number of strains tested. The serotypes defined by these three MAbs were also found in Italy. In Germany, the serotypes defined by MAbs S48-3-13 and S53-32 appear to be more common, whereas the S48-3-17 and S51-3 serotypes are found only sporadically. However, a large-scale study will be necessary to depict more precisely the prevalence and geographical spread of these and other serotypes. Thus, with the generation of more O-antigen-specific MAbs, it will be possible not only to complete a serotyping scheme for Acinetobacter, but also to further define the prevalence of Acinetobacter serotypes in clinical settings worldwide.