Bactericidal activity of three β-lactams alone or in combination with a β-lactamase inhibitor and two aminoglycosides against Klebsiella pneumoniae harboring extended-spectrum β-lactamases

Objective: To study the bactericidal activity of β-lactam antibiotics (imipenem, cefepime, cefpirome) alone or in combination with a β-lactamase inhibitor (sulbactam) in the presence or absence of aminoglycoside (amikacin or isepamicin) against Klebsiella pneumoniae strains producing extended-spectrum β-lactamases (ESBLs).
Methods: We characterized 10 strains by means of analytic isoelectric focusing and pulsed-field gel electrophoresis. The ESBLs produced by these strains were derived from either TEM (TEM-1, TEM-2) or SHV-1. The killing-curve method was used for this bacterial investigation. Bacteria (final inoculum 5×10 ⁵ CFU/mL) were incubated with antibiotics at clinical concentrations obtained in vivo.
Results: All the combinations with cefepime or cefpirome + sulbactam were bactericidal, with a 4 log₁₀ decrease being obtained within 6 h without regrowth at 24 h, whereas imipenem alone, and combinations, gave a bactericidal effect within 6 h. The two cephalosporins alone decreased the inoculum of 4 log₁₀ at 6 h but regrowth was observed at 24 h. When the aminoglycoside was added, this bactericidal effect was obtained within 3 h with amikacin and within 1 h with isepamicin.
Conclusions: Cefepime + sulbactam or cefpirome + sulbactarn may be an alternative to imipenem for the treatment of patients with ESBL-producing K. pneumoniae. Aminoglycosides are often associated in nosocomial infections due to ESBL-producing K. pneumoniae: isepamicin acted faster than amikacin, but both worked well. To conclude, it may be prudent to avoid extended-spectrum cephalosporins as single agent when treating serious infections due to ESBL-producing K. pneumoniae. Addition of a β-lactamase inhibitor such as sulbactam ± aminoglycoside is advisable to avoid failure of treatment.     

Evaluation of antimicrobial susceptibility of bacteria containing the qnr gene and FOX-5 β-lactamase by four automated systems

The accuracy and performance of four automated instruments (BD Phoenix, MicroScan WalkAway, VITEK-2 and Wider) were evaluated for susceptibility testing of fluoroquinolones and beta-lactams with four clinical isolates of Klebsiella pneumoniae and the corresponding Escherichia coli transconjugants containing a plasmid carrying the qnr gene and coding for FOX-5 production. No major or very major errors were detected with the MicroScan system. Many of the minor errors for both quinolones and beta-lactams clustered around the intermediate breakpoints.     

Extended-spectrum β-lactamase (ESBL) CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae in Sofia, Bulgaria

During a survey of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria in 2001-2002, three isolates from Sofia (two Escherichia coli, one Klebsiella pneumoniae) showed cefotaxime MICs that were decreased in the presence of clavulanate and were 2-8-fold higher than those of ceftazidime. Resistance was transferred to a sensitive recipient strain of E. coli. Both wild-type and transconjugant strains produced a cefotaxime-hydrolysing beta-lactamase of pI 8.8. Sequencing of the PCR product obtained with oligonucleotide primers binding outside the coding region identified this beta-lactamase as CTX-M-15. To our knowledge, this is the first report of CTX-M-15 in Bulgaria.     

Long-term study of the frequency of Escherichia coli and Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases

In total, 438 (1.7%) Escherichia coli and 125 (3.98%) Klebsiella pneumoniae isolates were found to be producers of extended-spectrum beta-lactamase (ESBL) during 1995-2003 in southern Spain. There was a significant increase in the frequency of ESBL-producing E. coli isolates, from < 0.36% before 1999 to 4.8% in 2003, while the frequency of ESBL-producing K. pneumoniae isolates decreased during the same period. The most common ESBLs detected in K. pneumoniae were SHV type, whereas both CTX-M and SHV types were detected in E. coli. In addition, E. coli isolates showed greater clonal diversity (84 distinct REP-PCR patterns, compared with five in K. pneumoniae), fewer enzymes per isolate, and a higher number of isolates recovered from outpatients. These differences may have implications for the control measures that should be used for these two microorganisms.     

Activity of ciprofloxacin and levofloxacin in experimental pneumonia caused by Klebsiella pneumoniae deficient in porins, expressing active efflux and producing QnrA1

The objective of this study was to evaluate the activities of ciprofloxacin and levofloxacin in a murine model of pneumonia caused by Klebsiella pneumoniae C2 (with altered GyrA, deficient in porins and expressing active efflux of quinolones) and the transconjugant C2pMG252 derived from it and expressing the qnrA1 determinant. MICs and MBCs of the two quinolones were determined according to CLSI guidelines. Time-kill curves (at 1× and 4× MIC) were also performed to assess bactericidal activity. An experimental model of pneumonia in mice was evaluated. Groups of 15 mice were infected with either strain and treated with ciprofloxacin (80 mg/kg/day) or levofloxacin (100 mg/kg/day). Control non-treated animals were also evaluated. In the case of strain C2, log₁₀ CFU/g of lung in non-treated animals was 9.16 ± 2.16. This value was reduced to 3.53 ± 1.04 (p <0.001) and 3.38 ± 0.46 (p <0.001) in animals treated with ciprofloxacin or levofloxacin, respectively. Percentages of surviving mice were 26.7% (control group) and 100% (both ciprofloxacin and levofloxacin; p <0.001 vs. controls). Bacterial counts (log₁₀ CFU/g) in lungs of animals infected with strain C2pMG252 were 9.65 ± 2.49 in non-treated animals and 7.74 ± 2.67 and 7.57 ± 3.84 for those treated with ciprofloxacin or levofloxacin, respectively (p >0.05 vs. control group). Of non-treated animals infected with strain C2pMG252, 14.3% survived. Ciprofloxacin and levofloxacin improved the survival in these mice (53.3% for both antimicrobials, p 0.03). In conclusion , the expression of qnrA1 in K. pneumoniae with additional mechanisms of resistance causes decreased efficacy of fluoroquinolones in a pneumonia model in mice.     

False susceptibility to cefotetan reported by MicroScan for DHA-type AmpC β-lactamase-producing Klebsiella pneumoniae

This study evaluated the accuracy of cefotetan susceptibility determination using the MicroScan WalkAway system for AmpC-producing Klebsiella pneumoniae. In total, 57 K. pneumoniae isolates that showed a D-shape flattening in a double-disk synergy test were studied. Cefotetan MICs were determined by the agar dilution method. The bla(DHA) gene was detected in all 57 isolates, one of which co-harboured bla(CMY-1). According to the MicroScan system, 28 isolates were susceptible, 18 were intermediately-resistant, and 11 were resistant to cefotetan. Compared with the agar dilution method, very major, minor and major error rates were 28.1% (16/57), 47.4% (27/57) and 1.8% (1/57), respectively.     

Activity of imipenem against VIM-1 metallo-β-lactamase-producing Klebsiella pneumoniae in the murine thigh infection model

The in-vivo activity of imipenem against VIM-1-producing Klebsiella pneumoniae (VPKP) was assessed in a thigh infection model in neutropenic mice. Animals were infected with three VPKP isolates (imipenem MICs 2, 4 and 32 mg/L, respectively) and a susceptible clinical isolate (MIC 0.125 mg/L) that did not produce any beta-lactamase with broad-spectrum activity. Bacterial density at the site of infection was determined after imipenem treatment (30 and 60 mg/kg every 2 h for 24 h). The log(10) reduction in CFU/thigh was greatest for the wild-type isolate, intermediate for the two imipenem-susceptible VPKP isolates, and lowest for the imipenem-resistant VPKP isolate. Whilst in-vivo imipenem activity appeared reduced against in-vitro susceptible VIM-1 producers compared with a VIM-1-negative control, an increased drug dosage could moderate this reduction.     

Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum β-lactamases

Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum beta-lactamases (ESBLs) were studied. Clinical records of patients with ESBL-positive K. pneumoniae isolates between 1989 and 2003 (n = 80) were reviewed retrospectively. Patients with SHV- and TEM-type ESBLs were identified more frequently in the intensive care units (67% and 78%, respectively), whereas those with CTX-M ESBLs were found in medical wards (52.2%) or were outpatients (17.4%) (p <0.01). The absence of urinary or central catheters was associated with CTX-M-10 (p 0.013 and p <0.01, respectively). Central catheter-related infections and secondary bacteraemia were associated more frequently with SHV- and TEM-type ESBLs, whereas urinary tract infections were associated with CTX-M-10. Previous aminoglycoside use was associated particularly with SHV-type ESBLs (p <0.01), whereas amoxycillin-clavulanate and oral cephalosporins were associated with CTX-M-10 (p <0.01 and p 0.050, respectively). The frequency of adequate empirical treatment was low (22%), and 61% of patients were treated according to the susceptibility testing results. Mortality (22%) and related mortality (14%) did not differ statistically according to the type of ESBL. Different ESBL types in K. pneumoniae were associated with different clinical variables, and this should be taken into account in current and future epidemiological scenarios.     

The first major extended-spectrum beta-lactamase outbreak in Scandinavia was caused by clonal spread of a multiresistant Klebsiella pneumoniae producing CTX-M-15

Between May and December 2005, 64 multidrug-resistant isolates of Klebsiella pneumoniae were detected from patients admitted to Uppsala University Hospital. This represented a dramatic increase in ESBL-producing K. pneumoniae compared to previous years. To investigate the epidemiology and to characterize the resistance mechanisms of the isolates, a study was initiated. Antibiotic susceptibility was determined by means of the Etest and the disc diffusion method. Extended-spectrum beta-lactamase (ESBL) production was identified by clavulanic acid synergy test and confirmed with PCR amplification followed by DNA sequencing. DNA profiles of the isolates were examined with pulsed-field gel electrophoresis (PFGE). All isolates were resistant or exhibited reduced susceptibility to cefadroxil, cefuroxime, cefotaxime, ceftazidime, aztreonam, piperacillin/tazobactam, ciprofloxacin, tobramycin, and trimethoprim-sulfamethoxazole. They produced ESBL of the CTX-M-15 type, and the involvement of a single K. pneumoniae clone was shown. This is the first major clonal outbreak of multiresistant ESBL-producing K. pneumoniae in Scandinavia. The outbreak demonstrates the epidemic potential of enterobacteria containing ESBLs of the CTX-M type, even in a country with a relatively low selective pressure and a low prevalence of multiresistant bacteria.     

Clonal dissemination of multilocus sequence type 11 Klebsiella pneumoniae carbapenemase – producing K. pneumoniae in a Chinese teaching hospital

Klebsiella pneumoniae carbapenemase (KPC)‐producing K. pneumoniae has disseminated rapidly in China. We aimed to analyze the molecular epidemiology of four KPC‐producing K. pneumoniae strains isolated from a suspected clonal outbreak during a 3‐month period and to track the dissemination of KPC‐producing K. pneumonia retrospectively. We created antimicrobial susceptibility profiles using an automated broth microdilution system and broth microdilution methods. We screened carbapenemase and KPC phenotypes using the modified Hodge test and meropenem–boronic acid (BA) disk test, respectively. We identified β‐lactamase genes with PCR and sequencing. We investigated clonal relatedness for epidemiological comparison using pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates expressed multidrug resistance and yielded positive results for the modified Hodge and meropenem‐BA disk tests. The isolates all carried bla_KPC‐2, and coproduced CTX‐M–type extended‐spectrum β‐lactamase. PFGE and MLST showed that the isolates were clonally related. The PFGE patterns of these isolates had ≥90% similarity. We found a single clone, sequence type (ST) 11, and its typical dissemination mode resembled clonal spread. The dissemination of KPC‐producing K. pneumoniae is clonally related and there is probable local transmission of a successful ST11 clone.     

白细胞介素22及其受体在大鼠肺炎克雷伯菌肺炎中的表达

目的 探讨白细胞介素22(IL-22)及IL-22受体IL-10R2在大鼠肺炎克雷伯菌肺炎发病过程中表达的意义。方法 50只SPF级SD大鼠按随机数字法分为肺炎组(n=25)和对照组(n=25)。肺炎组通过气管内注射肺炎克雷伯菌建立大鼠肺炎克雷伯菌肺炎模型,对照组则滴入等体积无菌生理盐水。造模后4h、1、2、3、4d通过观察大鼠的一般情况、肺组织大体和病理切片、肺组织细菌菌落计数对肺炎模型进行评价。采用实时荧光定量PCR和ELISA分别检测肺炎组和对照组大鼠造模后4h、1、2、3、4d肺组织IL-10R2 mRNA、外周血IL-22的表达水平。应用直线相关方法分析菌落计数与IL-22及其受体的相关性。结果肺炎组大鼠造模后4h出现活动度下降、聚集成团、竖毛现象;大体肺组织明显充血水肿,显微镜下肺泡结构破坏、肺泡腔及间质显著的充血及水肿、显著的中性粒细胞浸润。肺炎组大鼠肺组织均可培养出细菌,造模后4h细菌菌落计数最高[(0.94±0.02)×105 CFU/g],之后逐渐降低,至4d时降至最低[(0.07±0.00)×105 CFU/g],证实肺炎模型制备成功。与对照组比较,肺炎组IL- 10R2mRNA的表达量于造模后4h开始升高,于第3天达峰值（12.95±0.65比0.31±0.31,P＜0.05）,其后开始下降。肺炎组外周血中IL-22均低于同时间点对照组。肺炎组外周血中IL-22自造模后第3天较4h开始明显升高且随时间的延长逐渐升高。肺炎组中IL-10R2 mRNA、外周血IL-22的表达水平均与同时间点肺组织菌落计数呈负相关（r=-0.64,P=0.01;r=-0.59,P=0.00）。结论 IL-10R2在肺炎克雷伯菌肺炎中高表达,而外周血中IL-22可能由于消耗导致降低,但可随肺炎的好转回升。IL-22是肺炎克雷伯茵肺炎早期的炎性反应因子,在体内具有清除细菌的作用。     

Recommendation for treatment of severe infections caused by Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs)

Extended-spectrum β-lactamase (ESBL)-producing organisms are a global problem. No randomized controlled trials have ever been performed to guide optimal treatment. However, in vitro studies and observational studies strongly suggest that carbapenems (imipenem or meropenem) should be regarded as drugs of choice for serious infections due to ESBL-producing organisms. Other β-lactam antibiotics (cefepime, β-lactam/β-lactamase inhibitor combinations) are not suitable as first-line therapy. The increasing frequency of the association between quinolone resistance and ESBL production have greatly limited the role of this class of antibiotic against ESBL producers.     

Antimicrobial activity against strains of Escherichia coli and Klebsiella spp. with resistance phenotypes consistent with an extended-spectrum β-lactamase in Europe

Extended-spectrum β-lactamases (ESBLs) have continued to evolve after their initial detection in Europe nearly two decades ago. The summary results from the MYSTIC Program (31 medical centers) were utilized to assess the extent of ESBL occurrence in Europe from 1997 to 2000. ESBL phenotype rates in Klebsiella spp. (32.8%) and Escherichia coli (14.4%) were generally stable, but extensive hospital-to-hospital and unit-to-unit variations were noted. The highest ESBL rates were found in eastern Europe (including Turkey) and in intensive care unit patient populations. Carbapenems remained active against the ESBL-producing strains (meropenem MIC₉₀, 0.25–1 mg/L), while some other agents, such as aminoglycosides, fluoroquinolones, and piperacillin–tazobactam, were significantly less effective. International surveillance initiatives should be maintained to monitor future progression of this important resistance.     

CD4 T-lymphocyte subset counts in HIV-seropositive patients during the course of community-acquired pneumonia caused by Streptococcus pneumoniae

Total lymphocyte counts, CD4 T-lymphocyte counts and CD4/CD8 ratios were measured in 30 anti-retroviral-naive HIV-seropositive patients upon hospital admission for acute community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae, and again 1 month after resolution of infection. There was a significant depression of the total lymphocyte count (p < 0.005) and CD4 T-lymphocyte count (p < 0.001) in the acute stage of CAP caused by S. pneumoniae, with a subsequent increase in 90% (27/30) of cases after resolution of the infection. There was no significant difference in the CD4/CD8 T-lymphocyte ratio on admission compared with 1 month later (p 0.9).     

In vivo efficacy of cefotaxime and amoxicillin against penicillin-susceptible, penicillin-resistant and penicillin-cephalosporin-resistant strains of Streptococcus pneumoniae in a mouse pneumonia model

Objective: To compare cefotaxime (CTX) to amoxicillin (AMO) (usually considered the definitive therapy for penicillinsusceptible Streptococcus pneumoniae infections) in an immunocompromised mouse pneumonia model.
Methods: Three S. pneumoniae clinical isolates were used: two serotype 19 strains, a penicillin-susceptible (P^s) strain (penicillin MIC = 0.03 μ/mL) and a highly penicillin-resistant (P^r) strain (penicillin MIC = 4 μ/mL), and one serotype 23F strain, a penicillin-cephalosporin-resistant (CFTR) strain (CTX MIC = 4 μ/mL).
Results: CTX activity in this mouse model of pneumonia induced by the highly penicillin-resistant strain of S. pneumoniae was lower than expected from its low MIC against this organism. Furthermore, AMO had greater efficacy than CTX against a CFTR S. pneumoniae strain.
Conclusion: Our data suggest that there is no major difference in the in vivo efficacy of the two agents, cefotaxime and amoxicillin, against penicillin-resistant and penicillin-cephalosporin-resistant S. pneumoniae.