| Chapter 1:The characteristics of infection caused by Acinetobacter baumanniiBackground:Acinetobacter baumannii(A. baumannii) is a gram-negative, non-fermenting, aerobic coccobacillus, which could be widely detected in nature as well as in hospitals. A. baumannii is an opportunistic pathogen that can cause nosocomial infections especially in immune compromised patients. These bacteria possess the ability of quickly becoming multidrug-resistant owing to their extraordinary intrinsic resistance mechanisms. In recent years, it has attracted much attention and become a red alert pathogen.These resistance mechanisms include four classes of β-lactamases, methylation of 16S ribosomal RNA, decreased membrane permeability, up-regulation of efflux pump, chromosomal mutations, plasmid transport of resistance gene, etc. Thus, treatment options for highly resistant A. baumannii were strictly limited. A. baumannii causes ventilator-associated-pneumonia, sepsis, meningitis, skin and soft tissue infection, spondylitis, arthritis, as well as urinary tract infection, especially in immunocompromised residents in intensive care unit, which are associated with higher mortality rates, longer hospitalizations, and increased health care expenditures. Among those infections, pneumonia is most often seen. Being an opportunistic pathogen, A. baumannii infections mostly base on some severe diseases like trauma, tumor, acute respiratory distress syndrome, and severe empyrosis. There is no consensus on which of those risk factors is closely related to mortality.Being one of the most crucial third-tertiary hospitals in China, our hospital is responsible for the reception of severe patients transferred from lower level hospitals. Thus, A. baumannii infection has a relatively high morbidity in our hospital. However, there was a lack of clinical research on the characteristics of A. baumannii infections in our hospital. In the present study, we analyzed the population that was infected by A. baumannii, the situation of drug resistance of those representative strains, as well as the risk factors and prognosis of A. baumannii infection. We also analyzed the factors that were associated with the infection of highly resistant A. baumannii strains compared to common strains. The present study provided a general look on A. baumannii infection and evidences on the prevention and empirical treatment of A. baumannii infection. Besides, the clinical study raises many problems that remain to solve.MethodsThis study was carried out retrospectively through searching the intranet of our hospital. A total of 842 patients were enrolled into the study, which were all diagnosed as A. baumannii infection, from January 2014 to December 2015. The including criteria were as follows:positive bacterial culture, symptoms and signs of infection, imaging evidence of infection, and respective treatment. Those continuous variables were recorded as mean±standard deviation. Besides, t-test and chi-square-test were used to conduct the comparative analysis. P<0.05 was defined as statistical significance. SPSS 22.0 was applied for statistical analysis.Results842 patients were included, among which 628 were male patients and the remaining 214 were female patients.As for age, old people (age≥60yr) group occupied the highest percentage (62.47%), while the youth (age>1,≤14yr) group the lowest with a rate of 1.66%. We collected patients infected by A. baumannii of two years,409 patients from the year 2014 and 433 patients from the year 2015. In the year 2014, there was no obvious peak or trough. In 2015, there were two peaks, one from December 2014 to January 2015, and the other from March 2015 to July 2015.These 842 patients infected by A. baumannii were from forty departments in our hospital. The number of patients from ICU ward ranked first. The second place was occupied by sanitarian ward, followed by respiratory ICU, then by respiratory ward.Most of the bacterial strains were isolated from sputum. Accordingly, most patients acquired A. baumannii pneumonia.Five hundred and sixty patients isolated A. baumannii strains more than once. And those isolates from one patient may be different strains.The percentage of the representative isolates from 842 patients that were highly resistant towards antibiotics was 71.02%. The percentage of the representative isolates from 842 patients that remain susceptible towards tigecycline was 45.13%. The percentage of the representative isolates from 842 patients that were resistant towards cefoperazone/sulbactam was 38.96% in 2014 and 63.06% in 2015. The difference between those two years has statistical significance. The percentages of the representative isolates from 842 patients that were resistant towards imipenem were 85.82% in 2014 and 83.83% in 2015.The percentage of prior antibiotic therapy was 76.25%. More than a half (57.6%) of those all 842 patients acquired concurrent infection by other pathogens. The percentage of mechanical ventilation was 54.51%, from which 21.61% acquired tracheal intubation, and the rest 36.34% received tracheotomy. Forty-five percent of the 842 patients had trauma or received operation recently. Among all the patients, 36.34% had coronary artery disease (CAD),18.41% had diabetes mellitus (DM), and 11.05% had chronic obstructive pneumonia disease (COPD).As for prognosis of A. baumannii infection,8.67% of the 842 patients were cured, most of which were newborns. Sixty-four percent of all those patients were improved. And the crude mortality was 13.06%.The comparison study for highly resistance group and common resistance group presented that, people who were infected by highly resistant A. baumannii strains tend to had elder age, COPD, and CAD. Besides, patients who received mechanical ventilation, especially tracheotomy were inclined to be infected by highly resistant A. baumannii strains, the same with prior antibiotic therapy. And patients who were infected by highly resistant,A. baumannii strains resulted in longer hospitalization and higher crude mortality. Other factors, like gender, having DM, receiving tracheal intubation, recent trauma or operation, and co-infected with other pathogens, had no statistical significance between highly resistance group and common resistance group.Conclusion1. The majority of patients infected by A. baumannii were male and old people. There were nosocomial epidemics.2. A. baumannii infection was widespread and ICU wards rank first in patients’number. HAP was most often seen.3. A. baumannii strains were highly resistant towards tigecycline, cefoperazone/sulbactam, and imipenem.4. Elder age, COPD, CAD, mechanical ventilation, especially tracheotomy and prior antibiotic therapy increased the risk of infection by highly resistant strains.5. Patients infected by highly resistant A. baumannii strains resulted in longer hospitalization and higher crude mortality.Chapter 2:In vitro screen of antimicrobial options against extensively drug-resistant Acinetobacter baumanniiBackground:Acinetobacter baumannii (A. baumannii) is a gram-negative, non-fermenting, aerobic coccobacillus, which could be widely detected in nature as well as in hospitals. In recent years, A. baumannii has attracted much attention due to its ability to acquire resistance to multiple antimicrobial agents and A. baumannii has become one of the most frequent pathogens that cause nosocomial infection. Thus, researchers and clinicians paid much attention to A. baumannii infection in recent decades. A. baumannii causes ventilator-associated-pneumonia, sepsis, meningitis, skin and soft tissue infection, spondylitis, arthritis, as well as urinary tract infection, especially in immunocompromised residents in intensive care unit (ICU), which are associated with higher mortality rates, longer hospitalizations, and increased health care expenditures. Among those infections, pneumonia is most often seen.According to the capacity of resistance, A. baumannii has been defined as multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) strains. MDR was defined as resistant to at least one agent in more than three antimicrobial categories. While XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories). PDR was defined as resisant to all agents detected. It is currently of great concern for the emergence and spread of bacterial resistance to multiple antimicrobials and at the origin of harsh infections. This is especially true for nosocomial pathogens isolated from clinical samples, where these superbugs may compromise advanced medicine treatment, including surgery, organ transplantation, treatment of Acquired Immune Deficiency Syndrome (AIDS) and haematological patients, etc. Among the commonly identified MDR, XDR or even PDR pathogens, the lactose-non-fermenting Gram-negative pathogens A. baumannii and Pseudomonas aeruginosa have occupied an important place. These bacterial species possess the ability of quickly becoming multidrug-resistant owing to their extraordinary intrinsic resistance mechanisms. That is, as long as one agent is used to fight against MDR pathogen infection, resistance to this agent is developed fast. Sometimes the gene for resistance can spread among strains. These resistance mechanisms include four classes of β-lactamases, methylation of 16S ribosomal RNA, decreased membrane permeability, up-regulation of efflux pump, chromosomal mutations, plasmid transport of resistance gene, etc.For years, antimicrobials used in treating MDR A. baumannii infection have been limited to colistin, imipenem, and β-lactamase inhibitors. Since imipenem-resistant A. baumannii isolates are being detected more often throughout the world, scientists have been trying hard to develop new antibiotics and look back for help from old antimicrobial agents. In addition, drug combination may be a better alternative, and it offers many advantages over mono-therapy.We carried out three parts of research.Section 1Sulbactam and tigecycline are frequently used in clinical practices in China. Colistin, due to its obvious ototoxicity and nephrotoxicity, is rarely used as first-line antimicrobial agent now in our nation. While in western countries, colistin is still applied as a first-line antibiotic especially relating to A. baumannii infection. Rifampin is often used against no other infection but tuberculosis. There are increasing reports that certify the antimicrobial activities of those four agents against MDR pathogens. However, data were lack as for the antimicrobial activities of colistin, rifampin, sulbactam as well as tigecycline and their combination effects against XDR-A. baumannii. In the present study, we detected the in vitro antimicrobial activities of rifampin, colistin, sulbactam, and tigecycline alone and in combinations against XDR A. baumannii.Methods:XDR-A. baumannii strains were isolated from patients in three hospitals affiliated to Shandong University, from November 2012 to June 2013. Only one strain from each patient was included. VITEKⅡ microbial analysis instruments were used to obtain these XDR-A. baumannii isolates.All of the strains were evaluated by Kirby-Bauer (K-B) method as resistant to multiple antimicrobials, including aztreonam, piperacillin, ticarcillin/clavulanate, imipenem, ceftazidine, ciprofloxacin, levofloxacin, gentamicin, amikacin, tobramycin, sulfamethoxazole, tetracycline and ceftriaxone.As a result, a total of 25 strains were isolated, from which 21 were from sputum,2 from blood, and 2 from wound.Broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) for rifampin, colistin, sulbactam, and tigecycline against XDR-A. baumannii strains. The checkerboard microdilution method was used to determine the in vitro activities of potential therapeutic combinations of these four antimicrobial agents. Accordingly, the fractional inhibitory concentration (FIC) and FIC index (FICI) were calculated for each of the combinations.Additionally, the combination effects were calculated at sub-MIC levels.Results:1. When tested as single drugs:Rifampin:MICrange 0.5-8 ug/mL, MIC50 2 ug/mL, MIC90 8ug/mL, rate of susceptibility 44%;Colistin:MICrange 2-16 ug/mL, MIC50 2ug/mL, MIC90 8ug/mL, rate of susceptibility 88%;Sulbactam:MICrange 4-≥128 ug/mL, MIC50 32ug/mL, MIC90 64ug/mL,, rate of susceptibility 16%;Tygecycline:MICrange 1-4 ug/mL, MIC50 2ug/mL, MIC90 2ug/mL, rate of susceptibility 92%.2. FICIColistin/rifampin:rate of partial synergy 56%, rate of addition 36%, rate of indifference 8%, no synergy and antagonism.Colistin/sulbactam:rate of partial synergy 32%, rate of addition 24%, rate of indifference 44%, no synergy and antagonism.Colistin/tigecycline:rate of synergy 4%, rate of partial synergy 4%, rate of addition 36%, rate of indifference 56%, no antagonism.Rifampin/sulbactam:rate of synergy 4%, rate of partial synergy 32%, rate of addition 40%, rate of indifference 24%, no antagonism.Rifampin/tigecycline:rate of synergy 4%, rate of partial synergy 60%, rate of addition 16%, rate of indifference 20%, no antagonism.Sulbactam/tigecycline:rate of synergy 8%, rate of partial synergy 56%, rate of addition 20%, rate of indifference 16%, no antagonism.The combination of sulbactam/tigecycline showed good in vitro activities against XDR-A. baumannii isolates, followed by combinations of rifampin/tigecycline and colistin/rifampin. Colistin/tigecycline showed the highest rate of indifference. None of the six combinations showed antagonistic effect.3. Combination effects at sub-MIC levelsColistin:the average original MIC was 4ug/mL; when combined with rifampin at its 0.25MIC, colistin’s MIC decreased to 0.83ug/mL; when combined with rifampin at its 0.5MIC, colistin’s MIC decreased to 0.45ug/mL. When combined with tigecycline at its 0.25MIC, colistin’s MIC decreased to 1.66ug/mL; when combined with tigecycline at its 0.5MIC, colistin’s MIC decreased to 0.26ug/mL. When combined with sulbactam at its 0.25MIC, colistin’s MIC decreased to 1.74ug/mL; when combined with sulbactam at its 0.5MIC, colistin’s MIC decreased to 1.21ug/mL.Rifampin:the average original MIC was 3.38ug/mL; when combined with tigecycline at its 0.25MIC, rifampin’s MIC decreased to 0.72ug/mL; when combined with tigecycline at its 0.5MIC, rifampin’s MIC decreased to 0.2ug/mL. When combined with colistin at its 0.25MIC, rifampin’s MIC decreased to 0.67ug/mL; when combined with colistin at its 0.5MIC, rifampin’s MIC decreased to 0.25ug/mL. When combined with sulbactam at its 0.25MIC, rifampin’s MIC decreased to 1.95ug/mL; when combined with sulbactam at its 0.5MIC, rifampin’s MIC decreased to 1.72 ug/mL.Sulbactam:the average original MIC was 36.43ug/mL; when combined with tigecycline at its 0.25MIC, sulbactam’s MIC decreased to 22.43ug/mL; when combined with tigecycline at its 0.5MIC, sulbactam’s MIC decreased to 2.24ug/mL. When combined with colistin at its 0.25MIC, sulbactam’s MIC decreased to 24.29ug/mL; when combined with colistin at its 0.5MIC, sulbactam’s MIC decreased to 8.05ug/mL. When combined with rifampin at its 0.25MIC, sulbactam’s MIC decreased to 28.21ug/mL; when combined with rifampin at its 0.5MIC, sulbactam’s MIC decreased to 25.26ug/mL.Tigecycline:the average original MIC was 2.15ug/mL; when combined with rifampin at its 0.25MIC, tigecycline’s MIC decreased to 0.54ug/mL; when combined with rifampin at its 0.5MIC, tigecycline’s MIC decreased to 0.42ug/mL. When combined with sulbactam at its 0.25MIC, tigecycline’s MIC decreased to 0.70ug/mL; when combined with sulbactam at its 0.5MIC, tigecycline’s MIC decreased to 0.57ug/mL. When combined with colistin at its 0.25MIC, tigecycline’s MIC decreased to 0.84ug/mL; when combined with colistin at its 0.5MIC, tigecycline’s MIC decreased to 0.58ug/mL.Conclusions:1. Colistin and tigecycline had good bacteriostatic activity against XDR-A. baumannii, while rifampin was not as active against XDR-A. baumannii isolates. Sulbactam was least potent against XDR-A. baumannii isolates.2. When tested in combination, the combinations of colistin/rifampin, rifampin/sulbactam, rifampin/tigecycline, and sulbactam/tigecycline showed good in vitro activities against XDR-Ab isolates. More importantly, these combination regimens could exert addictive or partially-synergistic effects at the sub-MIC levels against XDR-A. baumannii strains.Section 2Fluoroquinolones which have broad-spectrum activity against both Gram-negative and -positive pathogens are commonly used antimicrobial agents. Nowadays, resistance to fluoroquinolones could be found in most nosocomial isolates of A. baumannii. Fluoroquinolones have thus become a less than ideal treatment for A. baumannii -related infection. Sitafloxacin, a new fluoroquinolone, has been shown to have good in vitro activity against pathogens resistant to other fluoroquinolones. The rate of carbapenem-resistant A. baumannii susceptibility to sitafloxacin was deemed acceptable by other reports. Nevertheless data testing the antimicrobial activities of sitafloxacin alone and in combination with other agents against XDR-A. baumannii is lacking. In the present study, we explored the in vitro antimicrobial activities of sitafloxacin alone and in combination with rifampin, colistin, sulbactam, and tigecycline against XDR-A. baumannii.Methods:Another array of XDR-A. baumannii strains were isolated from clinical specimens in four tertiary hospitals affiliated to Shandong University, from November 2013 to May 2014. Only one strain from each patient was included. VITEK II microbial analysis instruments were used to obtain these XDR-A. baumannii isolates.All of the strains were evaluated by Kirby-Bauer (K-B) method as resistant to multiple antimicrobials, including aztreonam, piperacillin, ticarcillin/clavulanate, imipenem, ceftazidine, ciprofloxacin, levofloxacin, gentamicin, amikacin, tobramycin, sulfamethoxazole, tetracycline and ceftriaxone.As a result a total of 24 strains were isolated, from which 21 were from sputum, 1 from blood,1 from cerebrospinal fluid, and 1 from urine.Broth microdilution assay was used to determine the MIC for sitafloxacin, rifampin, colistin, sulbactam, and tigecycline against XDR-A. baumannii strains. The checkerboard microdilution method was used to determine the in vitro activities of sitafloxacin combined with the other four antimicrobial agents. The FIC and FICI were calculated for each of the combinations. The decrease for average MIC values of one agent when combined with others at their 0.5MIC or 0.25MIC were also calculated.Results:1. When tested as single drugs:Sitafloxacin:MICrange 0.125-16ug/mL, MIC50 1ug/mL, MIC90 2ug/mL, rate of susceptibility 91.67%;Rifampin:MICrange 1-32 ug/mL, MIC50 4ug/mL, MIC9016ug/mL, rate of susceptibility 41.67%;Colistin:MICrange 0.5-64 ug/mL, MIC50 2ug/mL, MIC908ug/mL, rate of susceptibility 62.5%;Sulbactam:MICrange 4->128 ug/mL, MIC50 32ug/mL, MIC90 64 ug/mL, rate of susceptibility 16.67%;Tygecycline:MICrange 0.5-4 ug/mL, MIC50 2ug/mL, MIC90 4ug/mL, rate of susceptibility 54.17%.2. FICISulbactam/sitafloxacin:rate of synergy 12.5%, rate of partial synergy 41.67%, rate of addition 25%, rate of indifference 20.83%.Rifampin/sitafloxacin:rate of synergy 12.5%, rate of partial synergy 25%, rate of addition 29.17%, rate of indifference 33.33%.Colistin/sitafloxacin:rate of synergy 12.5%, rate of partial synergy 16.67%, rate of addition 20.83%, rate of indifference 50%. Tigecycline/sitafloxacin:rate of partial synergy 29.17%, rate of addition 41.67%, rate of indifference 29.17%.When tested in combination, all those three combinations except tigecycline/sitafloxacin revealed remarkable synergistic effects. Colistin/sitafloxacin showed the highest indifference rate. None of the four combinations showed antagonistic effect.3. Combination effects at sub-MIC levelsSitafloxacin:the average original MIC was 1.86ug/mL; when combined with sulbactam at its 0.25MIC, sitafloxacin’s MIC decreased to 1 ug/mL; when combined with sulbactam at its 0.5MIC, sitafloxacin’s MIC decreased to 0.76ug/mL. When combined with rifampin at its 0.25MIC, sitafloxacin’s MIC decreased to 1.37ug/mL; when combined with rifampin at its 0.5MIC, sitafloxacin’s MIC decreased to 0.78ug/mL. When combined with colistin at its 0.25MIC, sitafloxacin’s MIC decreased to 1.05ug/mL; when combined with colistin at its 0.5MIC, sitafloxacin’s MIC decreased to 1.04ug/mL.When combined with tigecycline at its 0.25MIC, sitafloxacin’s MIC decreased to 1.82ug/mL; when combined with tigecycline at its 0.5MIC, sitafloxacin’s MIC decreased to 1.15ug/mL.Sulbactam:the average original MIC was 29.54ug/mL; when combined with sitafloxacin at its 0.25MIC, sulbactam’s MIC decreased to 22.25ug/mL; when combined with sitafloxacin at its 0.5MIC, sulbactam’s MIC decreased to 12.79ug/mL.Rifampin:the average original MIC was 5ug/mL; when combined with sitafloxacin at its 0.25MIC, rifampin’s MIC decreased to 3.82ug/mL; when combined with sitafloxacin at its 0.5MIC, rifampin’s MIC decreased to 3.31ug/mL.Colistin:the average original MIC was 5.44ug/mL; when combined with sitafloxacin at its 0.25MIC, colistin’s MIC decreased to 4.16ug/mL; when combined with sitafloxacin at its 0.5MIC, colistin’s MIC decreased to 2.05ug/mL.Tigecycline:the average original MIC was 2.71ug/mL; when combined with sitafloxacin at its 0.25MIC, tigecycline’s MIC decreased to 2.2ug/mL; when combined with sitafloxacin at its 0.5MIC, tigecycline’s MIC decreased to 1.73ug/mL.Conclusions:1. Sitafloxacin has good in vitro activity against XDR-A. baumannii strains.2. The susceptibilities of colistin, rifampin and tigecycline against XDR-A. baumannii were decreased, especially that of tigecycline, in comparison with the results of Section 1.3. Compared with single drugs, most of the combinations of these antimicrobial agents could exert synergistic and/or partially synergistic and/or addictive effects, which might provide a better alternative when treating XDR-A. baumannii infections.Section 3In the last section, we concluded that sitafloxacin and sulbactam had good in vitro synergistic effect according to the results of FICI. To further testify the combination effect of sitafloxacin and sulbactam, we carried out the time-kill assay.XDR-A. baumannii strains were isolated from clinical specimens in three tertiary hospitals affiliated to Shandong University, from November 2014 to December 2015. For patients A. baumannii strains isolated more than once, only one strain from each patient was included. VITEKⅡ microbial analysis instruments were used to identify these XDR-A. baumannii isolates. Kirby-Bauer (K-B) method was applied to re-evaluate the strains to meet the criteria for XDR-A. baumannii. As a result, a total of fifty strains were included, of which thirty-six strains were from sputums, five from lavages, three from blood, three from wounds or surgical incisions, two from cerebrospinal fluid, and one from urine. ATCC25922 and ATCC27853 were used as quality control.Methods:Broth microdilution assay and Checkerboard microdilution assay were conducted as previous referring to section 1 and section 2.Time-kill assayTime-kill assay was conducted for four representative strains possessing different susceptibilities towards sitafloxacin and sulbactam. Bacterial suspensions were prepared following former steps and turbidities adjusted respectively. Thereafter, drugs alone or in combination were added into the suspensions and incubated at 37℃. Samples were removed at time point of 0,1,2,4,6,12 and 24 h. Aliquots (100 ul) were serially diluted in cold and sterile phosphate buffer saline. Bacterial counts were determined by plating three spots of 10 ul of appropriate dilutions on MH agar plates and incubating them at 35℃ for 18 to 24 h. Time-kill curves were then constructed by plotting mean colony counts (log10 CFU/ml) versus time. The bactericidal activities of drug combination were defined as a 3 log 10 CFU/ml (99.9%) reduction compared to gowth control at 24 h. Synergy was defined as a 2 log 10 CFU/ml decrease between the combination and the most active agent alone at 24 h. The drug combination was considered to be antagonistic with a≥2 log 10 increase in counts, and the combination was considered to be indifferent, if there was a< 2 log 10 increase or decrease in colony count with the combination when compared with the most active drug alone.The experiment was performed in duplicate to ensure reproducibility.Results:1. The MIC50 and MIC90 for sitafloxacin decreased and susceptibility rate increased when combined with sulbactam. The same for sulbactam as it combined with sitafloxacin.2. Sitafloxacin-sulbactam revealed synergistic effect in 16% of the fifty strains. The majority was constituted by partial synergy effect with a rate of 50%. None showed antagonistic effect.3. According to the susceptibility status of the fifty isolates toward sulbactam and sitafloxacin, they were organized into four groups.Seven isolates were susceptible to both sulbactam and sifafloxacin (SS).Thirty-nine isolates were sulbactam non-susceptible and sifafloxacin susceptible (NS).One isolate was sulbactam susceptible and sifafloxacin non-susceptible (SN). Three isolates were non-susceptible to either sulbactam or sifafloxacin (NN).4. As for bactericidal effects measured by time-kill assays, sitafloxacin revealed fast bacteriostatic effect at the concentration of 1 xMIC, regardless of the susceptibility status of the four isolates. When relating to sitafloxacin and sulbactam combination at the concentration of 0.5×MIC, bactericidal effect was achieved for the strains of SS and NS, and synergistic effect was revealed for all other strains but NN. When sitafloxacin and sulbactam combined at the concentration of 1 xMIC, the combination revealed bactericidal activity against all four isolates at 24h. Besides, synergistic effect was shown in all of the four isolates, regardless of the susceptibility status. What is more impressive, the strains of SS and NS were completely removed with no regrowth after 24h when sitafloxacin and sulbactam combined at the concentration of 1×MIC were applied.Conclusions:1. Sitafloxacin has good in vitro activity against XDR-A. baumannii strains.2. Sitafloxacin and sulbactam revealed bactericidal activity and synergistic effect. |
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