Evaluation of Gallium Citrate Formulations against a Multidrug-Resistant Strain of Klebsiella pneumoniae in a Murine Wound Model of Infection

Skin and soft tissue infections (SSTIs) are a common occurrence in health care facilities with a heightened risk for immunocompromised patients. Klebsiella pneumoniae has been increasingly implicated as the bacterial agent responsible for SSTIs, and treatment can be challenging as more strains become multidrug resistant (MDR). Therefore, new treatments are needed to counter this bacterial pathogen. Gallium complexes exhibit antimicrobial activity and are currently being evaluated as potential treatment for bacterial infections. In this study, we tested a topical formulation containing gallium citrate (GaCi) for the treatment of wounds infected with K. pneumoniae. First, the MIC against K. pneumoniae ranged from 0.125 to 2.0 μg/ml GaCi. After this in vitro efficacy was established, two topical formulations with GaCi (0.1% [wt/vol] and 0.3% [wt/vol]) were tested in a murine wound model of MDR K. pneumoniae infection. Gross pathology and histopathology revealed K. pneumoniae-infected wounds appeared to close faster with GaCi treatment and were accompanied by reduced inflammation compared to those of untreated controls. Similarly, quantitative indications of infection remediation, such as reduced weight loss and wound area, suggested that treatment improved outcomes compared to those of untreated controls. Bacterial burdens were measured 1 and 3 days following inoculation, and a 0.5 to 1.5 log reduction of CFU was observed. Lastly, upon scanning electron microscopy analysis, GaCi treatment appeared to prevent biofilm formation on dressings compared to those of untreated controls. These results suggest that with more preclinical testing, a topical application of GaCi may be a promising alternative treatment strategy for K. pneumoniae SSTI.     

Pharmacokinetics and Pharmacodynamics of Nemonoxacin against Streptococcus pneumoniae in an In Vitro Infection Model

The aim of this paper was to investigate the pharmacokinetics (PK) and pharmacodynamics (PD) of nemonoxacin, a novel nonfluorinated quinolone, against Streptococcus pneumoniae in vitro. A modified infection model was used to simulate the pharmacokinetics of nemonoxacin following scaling of single oral doses and multiple oral dosing. Four S. pneumoniae strains with different penicillin sensitivities were selected, and the drug efficacy was quantified by the change in log colony counts within 24 h. A sigmoid maximum-effect (E_max) model was used to analyze the relationship between PK/PD parameters and drug effect. Analysis indicated that the killing pattern of nemonoxacin shows a dualism which is mainly concentration dependent when the MIC is low and that the better PK/PD index should be the area under the concentration-time curve for the free, unbound fraction of the drug divided by the MIC (fAUC_0–24/MIC), which means that giving the total daily amount of drug as one dose is appropriate under those conditions. When the MIC is high, the time (T) dependency is important and the valid PK/PD index should be the cumulative percentage of a 24-h period in which the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (f%T>MIC), which means that to split the maximum daily dose into several separate doses will benefit the eradication of the bacteria. To obtain a 3-log₁₀-unit decrease, the target values of fAUC_0–24/MIC and f%T>MIC are 47.05 and 53.4%, respectively.     

Activity of Amoxicillin-Clavulanate against Penicillin-Resistant Streptococcus pneumoniae in an Experimental Respiratory Infection Model in Rats

High doses of amoxicillin, equivalent to those produced by 500- and 750-mg oral doses in humans (area under the plasma concentration-time curve), were effective against a penicillin-resistant strain of Streptococcus pneumoniae in an experimental respiratory tract infection in immunocompromised rats; this superior activity confirms the results of previous studies. An unexpected enhancement of amoxicillin’s antibacterial activity in vivo against penicillin-resistant and -susceptible S. pneumoniae strains was observed when subtherapeutic doses of amoxicillin were coadministered with the β-lactamase inhibitor potassium clavulanate. The reason for this enhancement was unclear since these organisms do not produce β-lactamase. The differential binding of clavulanic acid and amoxicillin to penicillin-binding proteins may have contributed to the observed effects.     

Emergence of Resistant Klebsiella pneumoniae in the Intestinal Tract during Successful Treatment of Klebsiella pneumoniae Lung Infection in Rats

Antibiotic treatment of lung infections may lead to the emergence of resistance in the gut flora. Appropriate dosing regimens could mitigate this adverse effect. In gnotobiotic rats harboring intestinal Escherichia coli and Enterococcus faecium populations, a lung infection by Klebsiella pneumoniae was instigated with two different sizes of inoculum to represent an early or a late initiation of antibiotic treatment. The rats were treated with marbofloxacin, an expanded-spectrum fluoroquinolone, by a single-shot administration or a fractionated regimen over 4 days. Intestinal bacterial populations were monitored during and after treatment. At the infection site, bacterial cure without any selection of resistance was observed. Whatever the dosage regimen, fluoroquinolone treatment had a transient negative impact on the E. coli gut population but not on that of E. faecium. The intestinal flora was colonized by the pathogenic lung bacteria, and there was the emergence of intestine-resistant K. pneumoniae, occurring more often in animals treated with a single marbofloxacin dose than with the fractionated dose. Bacterial cure without resistance selection at the infection site with fluoroquinolone treatment can be linked to colonization of the digestive tract by targeted pulmonary bacteria, followed by the emergence of resistance.The emergence of antimicrobial resistance during antibiotic treatment can occur in the infected organ system and/or in the endogenous normal gut flora (3). Antimicrobial agents, including fluoroquinolones, can be extensively excreted into the intestinal tract, exposing the normal host flora to antimicrobial selective pressure (i.e., inhibition of competing microflora). This may lead to a secondary development of antibiotic-resistant gut organisms (2, 3, 17). Klebsiella pneumoniae is an important opportunistic pathogen implicated in nosocomial bacterial infections (19). Epidemiological studies have shown that the majority of K. pneumoniae infections are often preceded by colonization of the patient''s gastrointestinal tract by the bacteria (9, 10). Recent reports suggest that fluoroquinolone-resistant Klebsiella pneumoniae isolates are common in many long-term care facilities and hospitals and are often associated with multidrug-resistant phenotypes (11, 22). The origins of this resistant K. pneumoniae gut subpopulation deserve attention. A possible factor contributing to the emergence of a resistant subpopulation of K. pneumoniae in the gut could be inadequate treatment of a prior K. pneumoniae infection, with secondary gut colonization by K. pneumoniae. This K. pneumoniae strain may then expand in the gut flora due to the selective pressure of the antibiotic reaching the gut lumen. Factors such as the concentration of the antimicrobial in the intestinal tract, the duration of the antimicrobial therapy, and the associated degree of disruption of the microflora may influence the likelihood that K. pneumoniae-resistant strains will or will not emerge at the gut level (3, 21). In previous studies on rodent models of Escherichia coli thigh infection and K. pneumoniae lung infection, we showed that the bacterial load at the start of antimicrobial treatment plays an important role in the enrichment of resistant strains at the infection site (4, 8). With a low inoculum, an early start of antimicrobial treatment with marbofloxacin, an expanded-spectrum fluoroquinolone extensively used in veterinary medicine, prevented mutant enrichment at the infection site, whereas a late start of the antimicrobial treatment with a high inoculum led to the enrichment of the resistant mutant subpopulation. Moreover, we showed that the emergence of resistance was dependent on the total marbofloxacin dose and dosage regimen.The aim of the present study was to assess, in a K. pneumoniae experimental infection model, the impact of different marbofloxacin dosage regimens on the commensal intestinal flora and to test the hypothesis that the critical site of emergence of resistance of a targeted lung pathogen during antibiotic treatment may be not the lung itself but the gut flora. With this as our aim, we developed a model of lung infection in gnotobiotic rats, with two inoculum sizes of K. pneumoniae, each treated with two different marbofloxacin dosage regimens. We chose to work with a gnotobiotic model harboring a Gram-positive and a Gram-negative bacterial population in the intestine.     

Carbapenem-Resistant Klebsiella pneumoniae Urinary Tract Infection following Solid Organ Transplantation

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging pathogen with a devastating impact on organ transplant recipients (OTRs). Data describing urinary tract infections (UTIs) due to CRKP, compared to extended-spectrum β-lactamase (ESBL)-producing and susceptible K. pneumoniae, are lacking. We conducted a retrospective cohort study comparing OTRs with a first episode of UTI due to CRKP, ESBL-producing K. pneumoniae, or susceptible K. pneumoniae. We identified 108 individuals; 22 (20%) had UTIs due to CRKP, 22 (20%) due to ESBL-producing K. pneumoniae, and 64 (60%) due to susceptible K. pneumoniae. Compared to susceptible K. pneumoniae (27%), patients with UTIs due to CRKP or ESBL-producing K. pneumoniae were more likely to have a ≥24-hour stay in the intensive care unit (ICU) before or after development of the UTI (64% and 77%, respectively; P < 0.001). Among 105/108 hospitalized patients (97%), the median lengths of stay prior to UTI with CRKP or ESBL-producing K. pneumoniae (7 and 8 days, respectively) were significantly longer than that for susceptible K. pneumoniae (1 day; P < 0.001). Clinical failure was observed for 8 patients (36%) with CRKP, 4 (18%) with ESBL-producing K. pneumoniae, and 9 (14%) with susceptible K. pneumoniae (P = 0.073). Microbiological failure was seen for 10 patients (45%) with CRKP, compared with 2 (9%) with ESBL-producing K. pneumoniae and 2 (3%) with susceptible K. pneumoniae (P < 0.001). In multivariable logistic regression analyses, CRKP was associated with greater odds of microbiological failure (versus ESBL-producing K. pneumoniae: odds ratio [OR], 9.36, 95% confidence interval [CI], 1.94 to 72.1; versus susceptible K. pneumoniae: OR, 31.4, 95% CI, 5.91 to 264). In conclusion, CRKP is associated with ICU admission, long length of stay, and microbiological failure among OTRs with UTIs. Greater numbers are needed to determine risk factors for infection and differences in meaningful endpoints associated with carbapenem resistance.     

Efficacy of Ceftaroline Fosamil against Escherichia coli and Klebsiella pneumoniae Strains in a Rabbit Meningitis Model

In this study, the efficacy of ceftaroline fosamil was compared with that of cefepime in an experimental rabbit meningitis model against two Gram-negative strains (Escherichia coli QK-9 and Klebsiella pneumoniae 1173687). The penetration of ceftaroline into inflamed and uninflamed meninges was also investigated. Both regimens were bactericidal, but ceftaroline fosamil was significantly superior to cefepime against K. pneumoniae and E. coli in this experimental rabbit meningitis model (P < 0.0007 against K. pneumoniae and P < 0.0016 against E. coli). The penetration of ceftaroline was approximately 15% into inflamed meninges and approximately 3% into uninflamed meninges.     

Treatment of Respiratory Klebsiella pneumoniae Infection in Mice with Aerosols of Kanamycin

Aerosols of kanamycin resulted in greater survival of mice challenged with respiratory Klebsiella pneumoniae than the same dosage given intramuscularly. Determinations of viable bacteria in the blood and lungs revealed that aerosolized kanamycin was most effective when infection was confined to the lungs. After the organisms had spread to other areas, however, aerosol therapy was still more effective than intramuscular therapy, but only one-half the infected mice survived.     

KPC酶在肺炎克雷伯菌碳青霉烯类耐药中的研究

目的运用RNA干扰技术探讨blaKPC表达与碳青霉烯类耐药的关系。方法用电转移法将双链小RNA（SiRNA）转入到2株产肺炎克雷伯菌碳青霉烯酶-2（KPC-2）的肺炎克雷伯菌中,检测RNA干扰前后菌株blaKPC的RNA水平和亚胺培南、厄他培南的最低抑菌浓度（MIC）值的变化。结果菌株在RNA干扰前后blaKPC-2的RNA水平有差异,但干扰前后亚胺培南和厄他培南的MIC值没有变化。结论 SiRNA虽然能在RNA水平上抑制blaKPC的表达,但对产KPC-2菌株碳青霉烯类耐药的影响不明显,证明革兰阴性杆菌对碳青霉烯类耐药由多种因素共同引起。     

KPC酶在肺炎克雷伯菌碳青霉烯类耐药中的研究

目的运用RNA干扰技术探讨blaKPC表达与碳青霉烯类耐药的关系。方法用电转移法将双链小RNA(SiRNA)转入到2株产肺炎克雷伯菌碳青霉烯酶-2(KPC-2)的肺炎克雷伯菌中,检测RNA干扰前后菌株blaKPC的RNA水平和亚胺培南、厄他培南的最低抑菌浓度(MIC)值的变化。结果菌株在RNA干扰前后blaKPC-2的RNA水平有差异,但干扰前后亚胺培南和厄他培南的MIC值没有变化。结论 SiRNA虽然能在RNA水平上抑制blaKPC的表达,但对产KPC-2菌株碳青霉烯类耐药的影响不明显,证明革兰阴性杆菌对碳青霉烯类耐药由多种因素共同引起。     

Activity of Antimicrobial Combinations against KPC-2-Producing Klebsiella pneumoniae in a Rat Model and Time-Kill Assay

This study evaluated the efficacy of tigecycline (TIG), polymyxin B (PMB), and meropenem (MER) in 80 rats challenged with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae infection. A time-kill assay was performed with the same strain. Triple therapy and PMB+TIG were synergistic, promoted 100% survival, and produced negative peritoneal cultures, while MER+TIG showed lower survival and higher culture positivity than other regimens (P = 0.018) and was antagonistic. In vivo and in vitro studies showed that combined regimens, except MER+TIG, were more effective than monotherapies for this KPC-producing strain.     

Comparative in vitro activities of temocillin and cefazolin against Klebsiella pneumoniae

Temocillin (BRL 17421) a new penicillin, was compared with cefazolin against 20 strains of Klebsiella. The in vitro minimal bactericidal concentration of cefazolin was twice as active as that of temocillin. Serum bactericidal levels obtained in volunteers treated with temocillin were consistently higher than those levels reached after the administration of cefazolin at the 6-h sampling.     

Activity of antibacterial impregnated central venous catheters against Klebsiella pneumoniae

OBJECTIVE: Antibiotically coated or impregnated catheters are effective in eliminating gram-positive bacteria from their surfaces. However, their activity against gram-negative bacteria is not well known. The aim of this study was to evaluate and compare the adherence, persistence and colonization of Klebsiella pneumoniae on catheter surfaces and also to assess bacteriostatic and bactericidal levels. DESIGN: Randomized, controlled, laboratory study. SETTING: University surgical microbiology laboratory. SUBJECTIVE: Silver sulfadiazine-chlorhexidine impregnated (SSC), minocycline and rifampin bonded (M+R), silver, platinum and carbon incorporated (SP+C) and non-antiseptic central venous catheter segments. INTERVENTIONS: Catheter segments were immersed in 1 ml of phosphate buffered saline (0.01 mol/l) with 0.25% dextrose (PBSD) and incubated at 37 degrees C. The PBSD was replaced daily. Effluents were frozen at -70 degrees C for subsequent determination of bacteriostatic and bactericidal activity. On days 1,3,7,14 and 21 after initial immersion, 1 ml standardized inoculum of Klebsiella pneumoniae was added to 90 tubes for a period of 30 min. The inoculum was then replaced with PBSD. One third of the samples were immediately sonicated and plated for the determination of bacterial adherence. The remaining segments were incubated for 4 and 24 h, followed by the same procedure to determine bacterial persistence and colonization with time. All plates were read after 24 h of incubation. MEASUREMENTS AND RESULTS: There was a significant reduction in initial bacterial adherence for SP+C catheters on all days ( p<0.05). SSC catheters prevented initial bacterial adherence for the first 7 days only ( p<0.05). SSC and SP+C catheters prevented bacterial persistence and further colonization on all days. However M+R catheters prevented bacterial colonization for 3 days only. Effluent studies indicated that the impregnated agents in catheter SSC were bactericidal compared to catheter M+R, which were bacteriostatic to K. pneumoniae. No antibacterial activity was detected in the effluents from catheter SP+C. CONCLUSIONS: SSC and SP+C catheters are effective in eliminating K. pneumoniae from their surfaces for at least 21 days. M+R catheters are less effective in eliminating bacterial adherence and colonization may be due to their bacteriostatic property.     

Activity of Moxifloxacin, Administered Once a Day, against Streptococcus pneumoniae in an In Vitro Pharmacodynamic Model of Infection

The antibacterial effect of moxifloxacin was studied by using an in vitro pharmacodynamic model of infection with dosing simulations of 400 mg every 24 h for 48 h. Streptococcus pneumoniae was tested by using four wild-type strains for which the moxifloxacin MICs were 0. 008, 0.12, 0.14, and 3.6 mg/liter. In addition, two isogenic mutants, generated from the strains for which the moxifloxacin MICs were /=1.0 mg/liter, little killing occurred over 48 h. A sigmoid dose-response model indicated that the area under the curve/MIC ratio was strongly related to the log change in viable count at 24 and 48 h and to the AUBKC. These data indicate that moxifloxacin may have a role in management of S. pneumoniae infection.     

Cytotoxic effect of green silver nanoparticles against ampicillin-resistant Klebsiella pneumoniae

Considering the harmful effects and high spread of drug-resistant Klebsiella pneumoniae, many researchers have been trying to produce new antibacterial agents to combat the emergence of multidrug-resistant (MDR) strains of this bacterium. Recent progress in the nanomedicine field has provided opportunities for synthesizing unique nanoagents to battle MDR bacteria by targeting virulence and resistance signalling. The biocidal effects of 14.9 nm silver nanoparticles fabricated using Nostoc sp. Bahar M (N-SNPs) and AgNO₃ were examined against drug-resistant K. pneumoniae using the agar well diffusion method. Transmission electron microscopy (TEM) was used to detect the ultrastructural changes caused by N-SNPs and AgNO₃. To address the mode of action of N-SNPs and AgNO₃, CAT, GPx, LDH and ATPase levels were assessed. The toxicity of N-SNPs and AgNO₃ was evaluated against the mfD, flu, hly, 23S, hns, hcp-1, VgrG-1 and VgrG-3 genes as well as cellular proteins. N-SNPs showed the greatest inhibitory activity against K. pneumoniae, with MIC and MBC values of 0.9 and 1.2 mg mL⁻¹, respectively. Furthermore, N-SNPs and AgNO₃ induced apoptotic features, including cell shrinkage and cell atrophy. N-SNPs were more potent bactericidal compounds than AgNO₃, causing increased leakage of LDH and GPx activities and depletion of ATPase and CAT activities, resulting in induced oxidative stress and metabolic toxicity. Compared to AgNO₃, N-SNPs exhibited the highest toxicity towards the selected genes and the greatest damage to bacterial proteins. N-SNPs were the most potent agents that induced bacterial membrane damage, oxidative stress and disruption of biomolecules such as DNA and proteins. N-SNPs may be used as effective nanodrugs against MDR bacteria.

Utilizing novel SNPs synthesized by Nostoc Bahar M sp. to combat the emergence of multidrug-resistant (MDR) strains of this bacterium through targeting virulence and resistance signalling.     

Evaluation of Low-Dose, Extended-Interval Clindamycin Regimens against Staphylococcus aureus and Streptococcus pneumoniae Using a Dynamic In Vitro Model of Infection

We have previously described the activity of low-dose clindamycin in extended-interval dosing regimens by determination of bactericidal titer in serum. In this study, we used a one-compartment in vitro dynamic infection model to compare the pharmacodynamics of clindamycin in three intravenous-dosing regimens (600 mg every 8 h [q8h], 300 mg q8h, and 300 mg q12h) against three clinical isolates of Staphylococcus aureus and two clinical isolates of Streptococcus pneumoniae. Test organisms were added to the central compartment of the model to yield a starting inoculum of 10(6) CFU/ml. Clindamycin was injected as a bolus into the central compartment at appropriate times over 48 h to simulate the q8h or q12h dosing regimens. Drug-free culture medium was then pumped through the system to mimic a half-life of 2.4 h. At predetermined time points during the experiment, samples were removed from the central compartments for colony count determination and drug concentration analysis. The rates of killing did not significantly differ among the three clindamycin dosing regimens against either S. aureus or S. pneumoniae (P = 0.88 or 0.998, respectively). Likewise, no significant differences in activities were detected among the three regimens against staphylococci (P = 0.677 and 0.667) or pneumococci (P = 0.88 and 0.99). Against an S. aureus isolate exhibiting inducible macrolide-lincosamide-streptogramin B resistance, none of the three clindamycin regimens prevented regrowth of the resistance phenotype in the model. In this model, clindamycin administered at a low dose in an extended-interval regimen (300 mg q12h) exhibited antibacterial activity equivalent to that of the 300- or 600-mg-q8h regimen.     

The effect of amikacin and imipenem alone and in combination against an extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strain

In vitro and in vivo activities of amikacin and imipenem alone, and in combination, were studied against an extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strain. The strain was in vitro susceptible to both antimicrobials at 10(5) and 10(7) CFU/mL. In time-kill studies amikacin, imipenem, and amikacin plus imipenem decreased the bacterial counts; difference between the bactericidal effects was not observed. Chequerboard technique showed no interaction between the tested drugs. Mice infected with 10(7) CFU/g of the K. pneumoniae were treated by amikacin (15 mg/kg every 8 h), imipenem (40 mg/kg every 4 h), or amikacin plus imipenem for 24 h. Blood bacterial counts in the group treated with amikacin plus imipenem did not differ significantly from the groups treated with amikacin or imipenem alone. Combination of amikacin and imipenem did not demonstrate any advantage over imipenem alone either in vitro or in vivo.     

Rapid Gentamicin Bioassay Using a Multiple-Antibiotic-Resistant Strain of Klebsiella pneumoniae

A rapid bioassay for gentamicin levels in serum was developed by using a strain of Klebsiella pneumoniae that has multiple resistance to antibiotics. Assays were comparable when performed on either small or large petri plates, and results were available in 2 to 4 h. Studies showed an overall recovery of 97.6% for gentamicin alone or 104.5% in the presence of commonly used antibiotics. The procedure can be performed without the need to inactivate other antibiotics that may be present in the serum sample.