Carbapenem-Resistant Acinetobacter baumannii in Three Tertiary Care Hospitals in Mexico: Virulence Profiles, Innate Immune Response and Clonal Dissemination

Abstract

Acinetobacter baumannii is one of the most important nosocomial pathogens distributed worldwide. Due to its multidrug-resistance and the propensity for the epidemic spread, the World Health Organization includes this bacterium as a priority health issue for development of new antibiotics. The aims of this study were to investigate the antimicrobial resistance profile, the clonal relatedness, the virulence profiles, the innate host immune response and the clonal dissemination of A. baumannii in Hospital Civil de Guadalajara (HCG), Hospital Regional General Ignacio Zaragoza (HRGIZ) and Pediatric ward of the Hospital General de México Eduardo Liceaga (HGM-P). A total of 252 A. baumannii clinical isolates were collected from patients with nosocomial infections in these hospitals between 2015 and 2016. These isolates showed a multidrug-resistant profile and most of them only susceptible to colistin. Furthermore, 83.3 and 36.9% of the isolates carried the bla _{OXA- 24} and bla _{TEM- 1} genes for resistance to carbapenems and β-lactam antibiotics, respectively. The clonal relatedness assessed by pulsed-field gel electrophoresis (PFGE) and by multi-locus sequence typing (MLST) demonstrated a genetic diversity. Remarkably, the ST136, ST208 and ST369 that belonged to the clonal complex CC92 and ST758 and ST1054 to the CC636 clonal complex were identified. The ST136 was a high-risk persistent clone involved in an outbreak at HCG and ST369 were related to the first carbapenem-resistant A. baumannii outbreak in HRGIZ. Up to 58% isolates were able to attach to A549 epithelial cells and 14.5% of them induced >50% of cytotoxicity. A549 cells infected with A. baumannii produced TNFα, IL-6 and IL-1β and the oxygen and nitrogen reactive species that contributes to the development of an inflammatory immune response. Up to 91.3% of clinical isolates were resistant to normal human serum activity. Finally, 98.5% of the clinical isolates were able to form biofilm over polystyrene tubes. In summary, these results demonstrate the increasingly dissemination of multidrug-resistant A. baumannii clones in three hospitals in Mexico carrying diverse bacterial virulence factors that could contribute to establishment of the innate immune response associated to the fatality risks in seriously ill patients.

Keywords: Acinetobacter baumannii; MLST; Mexico; adherence/invasion; biofilm; clonal dissemination; immune response; multidrug resistance.

FIGURE 1

Dendrogram generated from the analysis of 252 A. baumannii isolates from three tertiary care Mexican hospitals. A total of 202 isolates from HCG were grouped in 17 different clones, 42 isolates from HRGIZ were grouped in 12 different clones and 8 isolates from HGM-P were grouped in 4 different clones. Δ, φ: Isolates with similar PFGE profile disseminated in the three hospitals. One PFGE profile by clone was used to construct the dendrogram. The dashed line represents the 85% similarity level used in the cluster designation.

FIGURE 2

Members of A. baumannii clones identified during a 12-month study period. (A) Hospital Civil de Guadalajara HCG. (B) Hospital Regional General Ignacio Zaragoza, HRGIZ. (C) Pediatric ward, Hospital General de México Eduardo Liceaga HGM-P. The arrows show outbreaks of infections caused by A. baumannii.

FIGURE 3

eBURST analysis of A. baumannii isolated from three tertiary care hospitals in Mexico. Minimum spanning tree analysis showing the ST-groups of the MLST database where 5 STs of this study are located. (A) Related ST groups to ST136, ST369, and ST208 belonging to the clonal complex CC92. (B) Related ST groups to ST758 and 1054 belonging to the clonal complex CC636.

FIGURE 4

Biofilm production by isolates of A. baumannii. Each open circle corresponds to the average of two independent experiments, each one in triplicate (n = 6), plotted as the mean ± SD. (A) Bars indicate biofilm produced by A. baumannii ATCC-19606 by isolates from the HCG, HRGIZ and HGM-P. (B) Heat map chart indicates the level of biofilm production by every clinical isolate assessed. Every data indicate the biofilm index produced in relation to A. baumannii ATCC-19606. ATCC19606 A. baumannii was used as a control strain. ^∗p < 0.05; ^∗∗∗p < 0.001.

FIGURE 5

Resistance to normal human serum by isolates of A. baumannii. Percentage of bacterial survival is defined as % of serum resistance. Open circles correspond to the average of two independent experiments, each one in triplicate (n = 6) plotted as the mean ± SD. (A) Bars indicate serum resistance of A. baumannii ATCC-17961, S. typhi-9:12:Vid and by isolates from the HCG, HRGIZ, and HGM-P. (B) Heat map chart indicates the level of serum resistance of each clinical isolate assessed. A. baumannii-17961 was used as a strain with resistance to NHS. S. typhi was used as a strain with sensitivity to NHS. NS, non-significant.

FIGURE 6

Adherence/invasion of A. baumannii to A549 cells. Open circles correspond to each A. baumannii clinical isolate grouped by hospital in which was isolated each one in triplicate (n = 6). Each bar is plotted as the mean ± SD. (A) Bars indicate the adherence/invasion to A549 cells (relative index to A. baumannii-17961) of each isolate from the HCG, HRGIZ, and HGM-P. (B) Heat map chart indicates the level of adherence/invasion to A549 cells of each clinical isolate assessed. NS, non-significant; ^∗∗p < 0.01.

FIGURE 7

Cytotoxicity Induction by Isolates of A. baumannii on A549 cells. Each open circle corresponds to the average of two independent experiments in duplicated (n = 4). Each bar is plotted as the mean ± SD. (A) Bars indicate the cell death induction (cytotoxicity) of each isolate from the HCG, HRGIZ, and HGM-P on A549 cells. (B) Heat map chart indicates the level of cytotoxicity induced by each isolate on A549 cells. NS, non-significant; ^∗∗p < 0.01.

FIGURE 8

Production of TNFα, IL-6 and IL-1β by A549 infected-cells with isolates of A. baumannii. ELISA was used to quantify TNFα (A), IL-6 (B), and IL-1β (C) release. Each open circle corresponds to the average of two independent experiments, each one in duplicated (n = 4). Each bar is plotted as the mean ± SD. Bars in upper panels indicate the TNFα (A), IL-6 (B), and IL-1β (C) released by A549 cells infected with each isolate from the HCG, HRGIZ, and HGM-P. Heat map chart in lower panels indicates the level of cytokine released by A549 infected cells. NI, non-infected. NS, non-significant; ^∗p < 0.05.

FIGURE 9

Production of oxygen and nitrogen reactive species by A549 infected-cells with isolates of A. baumannii. The oxygen reactive species (superoxide anion) was quantified at 3 h post-infection (A) and the nitrogen reactive species (nitric oxide) at 24 h post-infection (B). Each open circle corresponds to the average of two independent experiments, each one in duplicated (n = 4). Each bar is plotted as the mean ± SD. Bars in upper panels indicate the superoxide anion (A) and nitric oxide (B) production by A549 cells infected with each isolate from the HCG, HRGIZ, and HGM-P. Heat map chart in lower panels indicates the level of superoxide anion and nitric oxide production by A549 infected cells. A. baumannii-17961 was used as a control strain of these experiments. NI, non-infected. NS, non-significant; ^∗p < 0.05.