Cluster Differences in Antibiotic Resistance, Biofilm Formation, Mobility, and Virulence of Clinical Enterobacter cloacae Complex

Abstract

Due to the lack of research on the characteristics of different clusters of Enterobacter cloacae complex (ECC), this study aimed to characterize and explore the differences among species of the ECC. An analysis based on hsp60 showed that Enterobacter hormaechei was predominant in ECC. Interestingly, the antibiotic resistance rates of clusters were different, among which E. hormaechei subsp. steigerwaltii (cluster VIII) and Enterobacter cloacae IX (cluster IX) possessed high resistant rates to ciprofloxacin and levofloxacin, but cluster II (Enterobacter kobei) had low resistant rates. Cluster II exhibited a strong biofilm formation ability. Different motility and protease production ability were shown for distinct clusters. A PCR analysis showed that clusters I, III, VI, VIII, and IX carried more virulence genes, while cluster II had fewer. Clusters I, VIII, and IX with high pathogenicity were evaluated using the Galleria mellonella infection model. Thus, the characteristics of resistance, biofilm-forming ability, mobility, and virulence differed among the clusters. The strains were divided into 12 subgroups based on hsp60. The main clusters of ECC clinical strains were I, II, III, VI, VIII, and IX, among which IX, VIII, and I were predominant with high resistance and pathogenicity, and cluster II (E. kobei) was a special taxon with a strong biofilm formation ability under nutrient deficiency, but was associated with low resistance, virulence, and pathogenicity. Hence, clinical classification methods to identify ECC subgroups are an urgent requirement to guide the treatment of clinical infections.

Keywords: Enterobacter cloacae complex; biofilm; clinical distribution; resistance; virulence.

FIGURE 1

An unrooted circle neighbor-joining tree with proportional branch length resulting from analysis of the hsp60 gene sequences of 130 clinical strains and previously reported sequences (Hoffmann and Roggenkamp, 2003; Hoffmann et al., 2005a,b; Iversen et al., 2008). The previously described strains correspond to sequences with GenBank accession numbers: E. asburiae ATCC 35853 T (AJ417141), E. kobei ATCC BAA260 T (AJ567899), E. cloacae subsp. dissolvens ATCC 23373 T (AJ417143), E. cloacae subsp. cloacae ATCC 13049 T (AJ417142), E. hormaechei subsp. steigerwaltii EN-562 T (AJ543908), E. hormaechei subsp. oharae EN-314 T (AJ543782), E. nimipressuralis ATCC 9912 T (AJ567900), E. hormaechei subsp. hormaechei ATCC 49162 T (AJ417108), E. ludwigii EN-119 T (AJ417114), E. cancerogenus ATCC 33241 T (AJ567895), E. amnigenus ATCC 3072 T (AJ567894), C. sakazaki ATCC 29544 T (AJ567902), E. cowanii ATCC 107300 T (AJ567896), E. pyrinus ATCC 49851 T (AJ567901), E. gergoviae ATCC 33028 T (AJ567897), and E. aerogenes AB008141 (AB008141). Genetic clusters are numbered according to the previous descriptions (Hoffmann and Roggenkamp, 2003). E., Enterobacter.

FIGURE 2

Distribution of clinical strains within the genetic clusters of ECC. All strains could be assigned to one of the previously reported hsp60 gene sequencing-based genetic clusters of ECC. (A) Distribution of ECC isolates in clusters. Colored square indicates the source of the ECC strain. (B) Cluster distribution numbers for ECC isolates at various anatomical sites. Colored square indicates the clusters of the ECC strain. ECC, Enterobacter cloacae complex.

FIGURE 3

Resistance of strains in six major clusters I, II, III, VI, VIII, and IX to commonly used clinical antibacterial drugs in ECC strains. Red squares indicate drug resistance, green squares indicate intermediation, and blue squares indicate sensitivity. One or more identical letters of a, b, c, and d in different cells indicate no statistical difference of resistance rate, while different letters of a, b, c, and d among the cells indicate a statistical difference. For example, for aztreonam, there is no difference in the resistance rate between I and VIII, II and IX, II and III, but a difference in resistance rate is observed between II and VI. ECC, Enterobacter cloacae complex; R, resistance; I, intermediation; S, sensitivity.

FIGURE 4

Comparison of the biofilm formation capacity of strains in clusters VIII, VI, III, II, I, and IX. *P < 0.05 means the biofilm formation capacity of clusters VIII, VI, III, I, and IX is significantly decreased compared to cluster II.

FIGURE 5

Bacterial mobility capacity of strains in clusters I, II, III, VI, VIII, and IX. *P < 0.05 means the swimming capacity of cluster I is significantly higher than clusters VI and VIII.

FIGURE 6

Protease production capacity of clusters I, II, III, VI, VIII, and IX. (A) The left panel shows the lysis loop of a high-protease-producing strain, the middle shows the lysis loop of a low-protease-producing strain, and the right shows no loop of a non-protease-producing strain. (B) The dots in the red box represent the protease-producing strains, and the dots outside the red box represent the non-protease-producing strains. *P < 0.05 means higher proteolytic activity of cluster IX compared to clusters II and III.

FIGURE 7

Different survival rates of G. mellonella infection model in clusters VIII, VI, III, II, I, and IX.