Genetic relatedness of third-generation cephalosporin-resistant Escherichia coli among livestock, farmers, and patients in Japan.

Objectives: The third-generation cephalosporin (3GC)-resistant E. coli strains have been detected worldwide in humans and animals. Hence, in this study, we evaluated the prevalence and genetic characteristics of 3GC-resistant E. coli in livestock, farmers, and patients to further analyse if livestock serves as a potential reservoir of antimicrobial-resistant bacteria. Methods: Faecal samples were collected from 330 healthy livestock (216 cattle and 114 swine), 61 healthy livestock farmers (52 cattle farmers and 9 swine farmers), and 68 non-duplicate 3GC-resistant E. coli isolates were also obtained from the clinical specimens of patients in Japan between 2013 and 2015. Genes associated with resistance in 3GC-resistant E. coli were identified using polymerase chain reaction (PCR) and DNA sequencing. Genotypic diversity was determined by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Results: We obtained 39 and 17 non-duplicated 3GC-resistant E. coli strains from healthy livestock (33 cattle and six swine) and livestock farmers, respectively. All isolates carried either CTX-M-type extended-spectrum ß-lactamase or plasmid-mediated AmpC ß-lactamase genes, with CTX-M-14 being the most frequent. CTX-M producers from livestock and patients belonged to 22 and 19 different sequence types (STs), respectively, and only three STs were the same. Among the 3GC-resistant E. coli from livestock and farmers, three types of CTX-M producers have shown similar characteristics (CTX-M genotype, ST, PFGE patterns, and antimicrobial susceptibilities) and were identified as clonal isolates shared among their farms. Conclusions: Our study findings indicate that CTX-M-14 is predominant in Japan. No distinct relationship was observed between the 3GC-resistant E. coli isolated from livestock and patients; however, some clonal relatedness was observed between the isolates from livestock and farmers due to their close contact.

Tigecycline Suppresses the Virulence Factors of Multidrug-Resistant Acinetobacter baumannii Allowing Human Neutrophils to Act.

Purpose: To determine the ability of human neutrophils to kill multidrug-resistant Acinetobacter baumannii (MDRAB) in the presence of tigecycline (TGC). Methods: Clinical isolates of MDRAB were cultured with human neutrophils and H2O2 in the presence of TGC. The numbers of viable bacteria, catalase activity, gene expression at the K locus of the MDRAB, reactive oxygen species (ROS) production, and granule exocytosis in human neutrophils were determined. Results: There was a time-dependent increase in the numbers of MDRAB after co-culturing with human neutrophils, whereas there was a significant decrease in the MDRAB numbers when co-cultured with both, human neutrophils and TGC for 6 h. The presence or absence of TGC did not affect total ROS production or the expression of CD11b, CD15, and CD63 on human neutrophils occurred when co-cultured with MDRAB. TGC significantly suppressed catalase activity and gene expression at the K locus of MDRAB, and significantly reduced the thickness of the capsule. Additionally, the bacterial viability of TGC-treated MDRAB cultured with H2O2 was lower than that without H2O2 after 6 h of culture. Conclusion: TGC significantly suppressed the expression of catalase and the capsule in MDRAB without adverse effects on neutrophil function, allowing human neutrophils to kill MDRAB. TGC is an effective antibiotic for treating MDRAB infections.

Effects of colistin and tigecycline on multidrug-resistant Acinetobacter baumannii biofilms: advantages and disadvantages of their combination.

We investigated the antimicrobial effects of colistin (CST) and tigecycline (TGC), either alone or in combination, on biofilm-dispersed and biofilm-embedded multidrug-resistant Acinetobacter baumannii (MDRAB) strains R1 and R2. The bacterial growth of biofilm-dispersed MDRAB was inhibited by CST or TGC. However, the inhibitory effects were attenuated by a combination of CST and low concentrations of TGC. The bactericidal effects of CST, but not TGC, were observed on biofilm-dispersed MDRAB. Notably, the bactericidal effects increased with a combination of CST and high concentrations of TGC, whereas they were attenuated with the combination of CST and low concentrations of TGC. Although biofilm formation by MDRAB decreased with increasing concentrations of CST or TGC, there was no complete disruption of the biofilms. Additionally, the biofilms increased with a combination of 1-2 µg/mL CST and TGC at 2 µg/mL and 2-4 µg/mL for strains R1 and R2, respectively. Biofilm-embedded MDRAB was eradicated with CST, but not TGC. Notably, the eradication effects increased with a combination of CST and high concentrations of TGC, whereas attenuation happened with the combination of CST and low concentrations of TGC. These results provide information on the combined effects of CST and TGC in the treatment of biofilm-associated MDRAB infection.

Histopathological Analysis of Acinetobacter baumannii Lung Infection in a Mouse Model.

Acinetobacter baumannii is the main causative pathogen of nosocomial infections that causes severe infections in the lungs. In this study, we analyzed the histopathological characteristics of lung infection with two strains of A. baumannii (ATCC 19606 and the clinical isolate TK1090) and Pseudomonas aeruginosa PAO-1 in C3H/HeN mice to evaluate the virulence of A. baumannii. Survival was evaluated over 14 days. At 1, 2, 5, or 14 days postinfection, mice of C3H/HeN were sacrificed, and histopathological analysis of lung specimens was also performed. Histopathological changes and accumulation of neutrophils and macrophages in the lungs after infection with A. baumannii and P. aeruginosa were analyzed. Following intratracheal inoculation, the lethality of ATCC 19606- and TK1090-infected mice was lower than that of PAO-1-infected mice. However, when mice were inoculated with a sub-lethal dose of A. baumannii, the lung bacterial burden remained in the mice until 14 days post-infection. Additionally, histopathological analysis revealed that macrophages infiltrated the lung foci of ATCC 19606-, TK1090-, and PAO-1-infected mice. Although neutrophils infiltrated the lung foci of ATCC 19606- and TK1090-infected mice, they poorly infiltrated the lung foci of PAO-1-infected mice. Accumulation of these cells in the lung foci of ATCC 19606- and TK1090-infected mice, but not PAO-1-infected mice, was observed for 14 days post-infection. These results suggest that A. baumannii is not completely eliminated despite the infiltration of immune cells in the lungs and that inflammation lasts for prolonged periods in the lungs. Further studies are required to understand the mechanism of A. baumannii infection, and novel drugs and vaccines should be developed to prevent A. baumannii infection.

Analysis of Immune Responses in Acinetobacter baumannii-Infected Klotho Knockout Mice: A Mouse Model of Acinetobacter baumannii Infection in Aged Hosts.

Acinetobacter baumannii is an important opportunistic pathogen that primarily afflicts elderly people. To clarify the pathogenicity of A. baumannii in the elderly, we investigated immune responses to A. baumannii ATCC 19606 infection in klotho knockout (KO) mice, the mouse model of aging. Following intravenous inoculation, the mice seldom displayed severe symptoms. However, the survival rate was 56% at 7 days post-infection. Bacteria were detected in the lungs of klotho KO mice but not klotho wildtype (WT) mice at 7 days post-infection. Neutrophils, eosinophils, interstitial macrophages, and monocyte/dendritic cell subset in the lungs of klotho KO mice were transiently induced after infection with A. baumannii. The number of alveolar macrophages in klotho KO mice was lower than that in klotho WT mice, except for 1 day post-infection. CD11b expression on neutrophils and alveolar macrophages in the lungs of klotho KO mice was seldom upregulated by the infection. These results suggested that immune functions eliminating bacteria in the lungs of klotho KO mice were insufficient. CD11blow conventional DC cells hardly increased in klotho KO mice infected with A. baumannii. Additionally, the production of interleukin (IL)-10 in the sera of klotho KO mice was significantly higher than that in klotho WT mice, whereas that production of interferon-gamma was not detected in the sera of klotho KO mice. These results suggested that acquired immune responses were hardly induced in klotho KO mice. IL-1ß, CXCL1, CXCL2, and CCL2 expression was significantly higher in the lungs of klotho KO mice infected with A. baumannii than in those of klotho WT mice at 1 day post-infection. These results suggested that pulmonary inflammation was elicited in klotho KO mice during early infection. The expression levels of proinflammatory cytokines significantly correlated with TLR9 expression in the lungs of klotho KO mice. The collective results demonstrate an A. baumannii infection state in aged hosts and suggest that pulmonary inflammation and bacterial burden should be noted in aged hosts even in the absence of severe symptoms of A. baumannii infection.

Lipopolysaccharide-Deficient Acinetobacter baumannii Due to Colistin Resistance Is Killed by Neutrophil-Produced Lysozyme.

Acinetobacter baumannii causes nosocomial infections due to its multidrug resistance and high environmental adaptability. Colistin is a polypeptide antibacterial agent that targets lipopolysaccharide (LPS) and is currently used to control serious multidrug-resistant Gram-negative bacterial infections, including those caused by A. baumannii. However, A. baumannii may acquire colistin resistance by losing their LPS. In mouse models, LPS-deficient A. baumannii have attenuated virulence. Nevertheless, the mechanism through which the pathogen is cleared by host immune cells is unknown. Here, we established colistin-resistant A. baumannii strains and analyzed possible mechanisms through which they are cleared by neutrophils. Colistin-resistant, LPS-deficient strains harbor mutations or insertion sequence (IS) in lpx genes, and introduction of intact lpx genes restored LPS deficiency. Analysis of interactions between these strains and neutrophils revealed that compared with wild type, LPS-deficient A. baumannii only weakly stimulated neutrophils, with consequent reduced levels of reactive oxygen species (ROS) and inflammatory cytokine production. Nonetheless, neutrophils preferentially killed LPS-deficient A. baumannii compared to wild-type strains. Moreover, LPS-deficient A. baumannii strains presented with increased sensitivities to antibacterial lysozyme and lactoferrin. We revealed that neutrophil-secreted lysozyme was the antimicrobial factor during clearance of LPS-deficient A. baumannii strains. These findings may inform the development of targeted therapeutics aimed to treat multidrug-resistant infections in immunocompromised patients who are unable to mount an appropriate cell-mediated immune response.

Resolvin E1, but not resolvins E2 and E3, promotes fMLF-induced ROS generation in human neutrophils.

E-series resolvins are biosynthesized from eicosapentaenoic acid during the resolution phase of acute inflammation and enhance inflammation resolution. However, the role of E-series resolvins in inflammation resolution is not yet known. Herein, we show that in human polymorphonuclear neutrophils (PMNs), resolvin E1 (RvE1) selectively enhances reactive oxygen species (ROS) generation induced by N-formylmethionyl-leucyl-phenylalanine. The RvE1-mediated enhancement is eliminated by a pan-antagonist of leukotriene B4 (LTB4) receptors, LY255283, or an NADPH oxidase inhibitor, diphenyleneiodonium. Thus, RvE1 enhances NADPH oxidase-mediated ROS generation via LTB4 receptors. Unlike RvE1, resolvins E2 and E3 do not show such activation of PMNs. Our findings suggest that RvE1 contributes to regulation of ROS generation, in accordance with the inflammatory state of the host.

Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion.

Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

The TNF-α of mast cells induces pro-inflammatory responses during infection with Acinetobacter baumannii.

Mast cells serve important roles as sentinels against bacterial infection by secreting mediators stored in granules. Much of their effectiveness depends upon recruiting and/or modulating other immune cells. The location of mast cells implies that they recognize pathogens invading tissues or mucosal tissues. Acinetobacter baumannii is a gram-negative bacterium that is considered an emerging nosocomial pathogen and causes a wide range of infections associated with high morbidity and mortality. To date, the interaction of A. baumannii with mast cells remains unclear. In this study, we demonstrated an interaction between human LAD2 mast cells and A. baumannii in vitro. When LAD2 cells were co-cultured with live A. baumannii or Pseudomonas aeruginosa PAO1 in vitro for 4h, TNF-α and IL-8 were produced in the culture supernatant. These inflammatory cytokines were not detected in the supernatant after the cells were treated with live bacteria without serum. Gene expression analysis showed that TNF-α and IL-8 mRNA expression increased in A. baumannii- and P. aeruginosa-infected LAD2 cells. Scanning electron microscopy showed that A. baumannii was tightly attached to the surface of LAD2 cells and suggested that A. baumannii may bind to FcγRII (CD32) on LAD2 cells. TNF-α in the culture supernatant from A. baumannii-infected LAD2 cells, showed that PMN activation and migration increased in Boyden chamber assays. These results suggest that mast cells recognize and initiate immune responses toward A. baumannii by releasing the preformed mediator TNF-α to activate effector neutrophils.

Spontaneous formation of neutrophil extracellular traps in serum-free culture conditions.

Neutrophils play a critical role in the innate immune response. Recently, a new neutrophilic biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. Neutrophils have been observed to release both lysosomal enzymes and their nuclear contents, including unfolded chromatin, which together trap and inactivate bacteria. The environment in tissues where neutrophils act is thought to be different from that of the blood serum. In this study, we assessed the effect of serum on NET formation. We found that neutrophils spontaneously form NETs in serum-free cultivation conditions at early times. These NETs functioned properly to trap bacteria. Furthermore, we demonstrated that reactive oxygen species play a critical role in the spontaneous formation of NETs. These results suggest that the serum condition must be considered in studies on neutrophils, including the formation and mechanism of action of NETs.

Inhibition of Neutrophil Adhesion and Antimicrobial Activity by Diluted Hydrosol Prepared from Rosa damascena.

Hydrosol prepared from the flowers of Rosa damascena (rose water) has been traditionally used for various health-related issues, including skin troubles such as erythema, itchiness, swelling. For the care of these skin troubles caused by microbial infection, both antimicrobial and antiinflammatory effects are required. Here, we investigated the effects of rose water on the growth of Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA), which cause skin infections, and on the function of neutrophils, which play a major role in the regulation of inflammatory reactions. To assess its modulatory effects on neutrophils, the effects of rose water against neutrophil adhesion response were evaluated. Rose water inhibited mycelial growth of C. albicans at a concentration of ca. 2.2%, and reduced viability of MRSA within 1 h. Rose water suppressed neutrophil activation induced by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), and N-formyl-Met-Leu-Phe (fMLP) at 5-15%. It also reduced the LPS- and TNF-α-induced cell surface expression of the adhesion-related molecule, cluster of differentiation (CD) 11b, but did not affect the migratory capacity of neutrophils with or without chemoattractant. These results suggest that rose water may reduce the pathogenicity of microbes, and attenuate neutrophil stimulation, which is involved in inflammatory responses. These findings suggest that rose water has a potential effect to inhibit skin inflammation caused by microbes.

A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8.

Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target.

Loop-mediated isothermal amplification: Rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii, which are mainly induced by the production of OXA-type ß-lactamases, are among the leading causes of nosocomial infections worldwide. Among the ß-lactamase genes, the presence of the OXA-51-like gene carrying the upstream insertion sequence, ISAba1, was found to be one of the most prevalent carbapenem resistance mechanisms utilized by these bacteria. Consequently, it is necessary to develop a rapid detection method for ISAba1-blaOXA-51-like sequence for the timely and appropriate antibiotic treatment of A. baumannii infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was optimized for ISAba1-blaOXA-51-like detection. The LAMP primer set was designed to recognize distinct sequences in the ISAba1-blaOXA-51-like gene and could amplify the gene within 25 min at an isothermal temperature of 60°C. This LAMP assay was able to detect the ISAba1-blaOXA-51-like gene with high specificity; in addition, no cross-reactivity was observed for other types of ß-lactamase producers (OXA-23-like, OXA-40-like, OXA-58-like, and IMP-1), as indicated by the absence of false positive or false negative results. The detection limit for this assay was found to be 10(0)CFU per tube which was 100-fold more sensitive than a polymerase chain reaction assay for ISAba1-blaOXA-51-like detection. Furthermore, the LAMP assay provided swift detection of the ISAba1-blaOXA-51-like gene, even directly from clinical specimens. In summary, we have described a new, rapid assay for the detection of the ISAba1-blaOXA-51-like gene from A. baumannii that could be useful in a clinical setting. This method might facilitate epidemiological studies and allow monitoring of the emergence of drug resistant strains.

Acinetobacter baumannii escape from neutrophil extracellular traps (NETs).

Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are usually harmless but cause infectious diseases in compromised hosts. Neutrophils play a critical role in infective protection against the extracellular growth of bacteria. Recently, a new biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention. In present study, we investigated the responsiveness of neutrophils to A. baumannii and P. aeruginosa, focusing on NET formation. Neutrophils were co-cultured with A. baumannii or P. aeruginosa, and then DNA, histone and neutrophil elastase were stained, and the formation of NETs was evaluated. Neutrophils stimulated with A. baumannii had spread, but their shapes was maintained, and the nucleus was observed as clearly as that in non-stimulated neutrophils. However, neutrophils stimulated with P. aeruginosa did not maintain their cellular morphology, and the nucleus was disrupted with DNA, histones, and neutrophil elastase released into the extracellular space. These results suggest that A. baumannii does not induce NET formation, in contrast to P. aeruginosa. In addition, we measured expression of myeloperoxidase (MPO), reactive oxygen species (ROS) and superoxide in neutrophils, and we found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated neutrophils. Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa. In this study, we show that the reactivity of neutrophils and their biological defense mechanism are different between A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria.

Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of ß-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae.

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.

Effects of erythromycin and rifampicin on immunomodulatory gene expression and cellular function in human polymorphonuclear leukocytes.

BACKGROUND: We investigated the effects of two antibiotics, erythromycin and rifampicin, on the immunomodulatory gene expression and cellular function of human polymorphonuclear leukocytes (PMNs). METHODS: We used real-time quantitative PCR to examine the expression of immunomodulatory genes. The production of reactive oxygen species (ROS) was determined by fluorescence-activated cell sorting. PMN chemotaxis was analyzed using a KK chemotaxis chamber. RESULTS: Stimulation of PMNs with lipopolysaccharide (LPS) resulted in increases in the mRNA levels of immunomodulatory genes. Rifampicin significantly inhibited the overexpression of TLR2, TLR4, CD14 and IL8Rs. However, erythromycin suppressed only the upregulation of TLR2 and TNFA. Neither antibiotic had an effect on the production of ROS. Rifampicin significantly inhibited PMN chemotaxis, but erythromycin had no effect. CONCLUSIONS: Erythromycin and rifampicin may play anti-inflammatory roles by affecting the expression levels of immunomodulatory genes or the chemotaxis of PMNs.

Analysis of membrane antigens on neutrophils from patients with sepsis.

The aim of the present study was to assess changes of cell membrane antigens on neutrophils in septic patients. Expression levels of neutrophil membrane antigens were measured employing a FACS calibur flow cytometer with several fluorescence-labeled monoclonal antibodies. Expression levels of the CD14 antigen were higher in patients with sepsis than in healthy individuals. In particular, the expression levels of CD14 increased in patients complicated by septic shock. Expression levels of TLR-4 were higher in patients with sepsis or septic shock than in healthy individuals. Expression levels of CD11b and CD16 were lower in patients with sepsis or septic shock than in healthy individuals and were even lower in those complicated by septic shock. Expression levels of neutrophil membrane antigens in patients with sepsis markedly changed in the acute phase. However, these levels tended to return to those of healthy individuals in the convalescing phase. Analyses of the surface antigens on neutrophils strongly involved in biological defense or tissue injury are informative for understanding the pathology of sepsis and for conducting therapy targeting neutrophils in the future.