Showing papers on "Klebsiella pneumoniae published in 2010"

Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum β-lactamase producing, Enterobacteriaceae infections: a systematic review

Abstract: Summary Rising rates of resistance to antimicrobial drugs among Enterobacteriaceae limit the choice of reliably active forms of these drugs. We evaluated the evidence on fosfomycin as a treatment option for infections caused by members of the family Enterobacteriaceae with advanced resistance to antimicrobial drugs, including producers of extended-spectrum β-lactamase (ESBL). We systematically reviewed studies evaluating the antimicrobial activity, or the clinical effectiveness of fosfomycin. 17 antimicrobial-susceptibility studies were found and included in our Review, accounting for 5057 clinical isolates of Enterobacteriaceae with advanced resistance to antimicrobial drugs (4448 were producers of ESBL); 11 of the 17 studies reported that at least 90% of the isolates were susceptible to fosfomycin. Using a provisional minimum inhibitory concentration susceptibility breakpoint of 64 mg/L or less, 1604 (96·8%) of 1657 Escherichia coli isolates producing ESBL were susceptible to fosfomycin. Similarly, 608 (81·3%) of 748 Klebsiella pneumoniae isolates producing ESBL were susceptible to fosfomycin. In two clinical studies, oral treatment with fosfomycin–trometamol was clinically effective against complicated or uncomplicated lower urinary tract infections caused by ESBL-producing E coli in, cumulatively, 75 (93·8%) of the 80 patients evaluated. Initial clinical data support the use of fosfomycin for the treatment of urinary tract infections caused by these pathogens, although further research is needed.

Klebsiella pneumoniae AcrAB Efflux Pump Contributes to Antimicrobial Resistance and Virulence

Abstract: Respiratory infections caused by Klebsiella pneumoniae are characterized by high rates of mortality and morbidity. Management of these infections is often difficult, due to the high frequency of strains that are resistant to multiple antimicrobial agents. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens. In the present study, we investigated the role of the K. pneumoniae AcrRAB operon in antimicrobial resistance and virulence by using isogenic knockouts deficient in the AcrB component and the AcrR repressor, both derived from the virulent strain 52145R. We demonstrated that the AcrB knockout was more susceptible, not only to quinolones, but also to other antimicrobial agents, including β-lactams, than the wild-type strain and the AcrR knockout. We further showed that the AcrB knockout was more susceptible to antimicrobial agents present in human bronchoalveolar lavage fluid and to human antimicrobial peptides than the wild-type strain and the AcrR knockout. Finally, the AcrB knockout exhibited a reduced capacity to cause pneumonia in a murine model, in contrast to the wild-type strain. The results of this study suggest that, in addition to contributing to the multidrug resistance phenotype, the AcrAB efflux pump may represent a novel virulence factor required for K. pneumoniae to resist innate immune defense mechanisms of the lung, thus facilitating the onset of pneumonia.

Worldwide diversity of Klebsiella pneumoniae that produce beta-lactamase blaKPC-2 gene.

Abstract: Klebsiella pneumoniaeisolates that produce carbapenemases (KPCs) are rapidly disseminating worldwide. To determine their genetic background, we investigated 16 blaKPC-2-harboring K. pneumoniae isolates from 5 countries. The isolates were multidrug resistant, possessed the blaKPC-2 gene, and differed by additional Beta-lactamase content. They harbored a naturally chromosome-encoded bla gene (blaSHV-1 [12.5%], blaSHV-11 [68.7%], or blaOKP-AVB [18.8%]) and several acquired and plasmid-encoded genes (blaTEM-1 [81.3%], blaCTX-M-2 [31.3%], blaCTX-M-12 [12.5%], blaCTX-M-15 [18.7%], and blaOXA-9 [37.5%]). The blaKPC-2 gene was always associated with 1 of the Tn4401 isoforms (a, b, or c). Tn4401 was inserted on different-sized plasmids that belonged to different incompatibility groups. Several blaKPC-containing K. pneumoniae clones were found: 9 different pulsotypes with 1 major (sequence type 258) and 7 minor distinct allelic profiles. Different clones harboring different plasmids but having identical genetic structure, Tn4401, could be at the origin of the worldwide spread of this emerging resistance gene.

An Outbreak of Infection due to β-Lactamase Klebsiella pneumoniae Carbapenemase 2–Producing K. pneumoniae in a Greek University Hospital: Molecular Characterization, Epidemiology, and Outcomes

Abstract: Background We describe the emergence and spread of Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing K. pneumoniae at a Greek University hospital. Methods Isolates with a carbapenem minimum inhibitory concentration >1 microg/mL and a negative EDTA-imipenem disk synergy test result were submitted to boronic acid disk test and to polymerase chain reaction (PCR) for KPC gene and sequencing. Records from patients who had KPC-2-producing K. pneumoniae isolated were retrospectively reviewed. Clinical isolates were submitted to molecular typing using pulsed-field gel electrophoresis, and the beta-lactamase content was studied using isoelectric focusing and PCR. Results From January 2007 through December 2008, 50 patients (34 in the intensive care unit [ICU]) were colonized (n = 32) or infected (n = 18) by KPC-2-producing K. pneumoniae. Increasing prevalence of KPC-2-producing K. pneumoniae coincided with decreasing prevalence of metallo-beta lactamase-producing isolates in our ICU. Multidrug resistance characterized the studied isolates, with colistin, gentamicin, and fosfomycin being the most active agents. Besides KPC-2, clinical isolates encoded TEM-1-like, SHV-11, SHV-12, CTX-M-15, and LEN-19 enzymes. Four different clonal types were detected; the predominant one comprised 41 single patient isolates (82%). Sporadic multiclonal cases of KPC-2-producing K. pneumoniae infection were identified from September 2007 through May 2008. The outbreak strain was introduced in February 2008 and disseminated rapidly by cross-transmission; 38 patients (76%) were identified after August 2008. Fourteen cases of bacteremia, 2 surgical site infections, 2 lower respiratory tract infections (1 bacteremic), and 1 urinary tract infection were identified. Most patients received a colistin-containing combination treatment. Crude mortality was 58.8% among ICU patients and 37.5% among non-ICU patients, but attributable mortality was 22.2% and 33.3%, respectively. Conclusions The emergence of KPC-2-producing K. pneumoniae in Greek hospitals creates an important challenge for clinicians and hospital epidemiologists, because it is added to the already high burden of antimicrobial resistance.

Synthesis and Spectrum of the Neoglycoside ACHN-490

Abstract: Aminoglycosides (AGs) are highly potent, broad-spectrum antibiotics used for treating serious bacterial infections. They are a well-established class of antibacterials that act by binding to the A-site of the bacterial ribosome and interfering with normal protein synthesis. They are frequently used empirically for treating complicated urinary tract infections, nosocomial respiratory tract infections, complicated intra-abdominal infections, and septicemia (4). AGs and β-lactam antibiotics are sometimes used in combination for treating suspected or confirmed Pseudomonas aeruginosa infections, as well as empirically in endocarditis. As seen with other antibiotic classes, resistance to AGs has emerged in the 50 years since their introduction. For example, gentamicin (GEN) resistance (MIC, >4 μg/ml) was seen in 31.4% of P. aeruginosa isolates from urinary tract infections collected in Europe and the Americas in 2000 (SENTRY Antimicrobial Surveillance Program) (12). Amikacin (AMK) resistance (MIC, >16 μg/ml) was seen in 16.7% of the same isolates. Enterobacteriaceae have also shown resistance to AMK (9). Among isolates collected from intra-abdominal infections worldwide in 2004 (Study for Monitoring Antibiotic Resistance Trends [SMART]), 9.0% of extended-spectrum β-lactamase-positive (ESBL+) Escherichia coli and 31.5% of ESBL+ Klebsiella pneumoniae were resistant to AMK (19). Other AGs were not examined in that study. At present, the most significant contribution to clinical resistance is represented by diverse AG-modifying enzymes (AMEs) (Fig. ​(Fig.1)1) that inactivate AGs by N-acetylation (AG acetyltransferases [AAC]), O-adenylylation (AG nucleotidyltransferases [ANT]), or O-phosphorylation (AG phosphotransferases [APH]) (11). Numerous such enzymes have been identified, and they often occur in combinations that can impart broad AG resistance (15). Less common AG resistance mechanisms (AGRMs) among the Enterobacteriaceae involve the regulation of intracellular drug concentration by overexpression of efflux pumps and the expression of enzymes that modify the ribosomal target (10). As broad-spectrum antibiotics, including the existing AGs cephalosporins, carbapenems, and fluoroquinolones, are rendered ineffective by increasing resistance, new AGs are an attractive option for the treatment of serious infections, especially if developed with optimized dosing regimens to maximize safety and efficacy (7). FIG. 1. ACHN-490 structure and AMEs from Gram-negative and Gram-positive (underlined) organisms. AMEs shown with dotted arrows cannot modify ACHN-490. In the present study, we modified sisomicin (SIS) in search of substituents that would improve the in vitro potency against AG-resistant (AG-R) bacterial strains. Among the SIS derivatives we synthesized, the novel compound ACHN-490 showed promise in a screening panel and thus was tested against a larger panel that included 461 recent clinical isolates of Gram-negative and Gram-positive organisms. The majority of the pathogens in these panels carried AGRMs, and many were also resistant to other classes of antibiotics. The broad-spectrum in vitro activity demonstrated in these experiments supports the continued development of ACHN-490. (These data were presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, September 2009 [1].)

Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results

Abstract: Objectives The aim of this study was to evaluate the presence of carbapenemases in a Klebsiella pneumoniae collection and the performance of the modified Hodge test (MHT) to correctly identify this phenotype. Methods Twenty-eight K. pneumoniae clinical isolates with reduced susceptibility to carbapenems were evaluated. Antimicrobial susceptibility and molecular typing were performed by agar dilution and PFGE, respectively. The MHT was performed using both standard and high inoculum of test organisms. Imipenem hydrolysis was investigated by spectrophotometric assays and carbapenemase-encoding genes were identified by PCR and amplicon sequencing. Porin loss was investigated by both PCR and SDS-PAGE. Results Susceptibility rates for imipenem, meropenem and ertapenem were 93%, 57% and 11%, respectively. The PFGE analysis showed seven unrelated genotypes. By testing standard inoculum and ertapenem or meropenem discs, 25% (n = 7) and 21% (n = 6) of the isolates were classified as carbapenemase producers, respectively. When a higher inoculum was employed, these rates increased to 54% (n = 15) and 43% (n = 12), respectively. No imipenem hydrolysis was detected. PCRs identified bla(CTX-M) in 27 (96%) isolates, of which 2 isolates also carried bla(GES-1.) SDS-PAGE and PCR assays revealed that all isolates had lost at least one outer membrane protein, except for a single isolate that was found to express both OmpK35 and OmpK36. Conclusions False detection of carbapenemase production was observed by the MHT possibly as a result of extended-spectrum beta-lactamase (ESBL) production coupled with porin loss as reported before. Clinical laboratories must be aware of this fact, especially in geographical areas where ESBL-producing isolates are highly prevalent.

Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination

Abstract: Background: Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. Methods: Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequencebased PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. Results: Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained blaOXA-23, and four isolates possessed blaOXA-24/40. Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing blaKPC-2 or blaKPC-3 were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). Conclusion: In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrugresistant phenotypes.

Role of type 1 and type 3 fimbriae in Klebsiella pneumoniae biofilm formation

Abstract: Background Klebsiella pneumoniae is an important gram-negative opportunistic pathogen causing primarily urinary tract infections, respiratory infections, and bacteraemia. The ability of bacteria to form biofilms on medical devices, e.g. catheters, has a major role in development of many nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. In this study, we characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation.

Bloodstream Infections Caused by Metallo-β-Lactamase/ Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae among Intensive Care Unit Patients in Greece: Risk Factors for Infection and Impact of Type of Resistance on Outcomes

Abstract: Objective. To determine risk factors for bloodstream infections (BSIs) caused by Klebsiella pneumoniae producing metallo–β‐lactamases (MBLs) or K. pneumoniae carbapenemases (KPCs), as well as risk factors for mortality associated with carbapenem‐resistant K. pneumoniae, among intensive care unit (ICU) patients. Methods. Two case‐control studies were conducted in a patient cohort with K. pneumoniae BSIs in an 8‐bed ICU in a Greek hospital from January 1, 2007, through December 31, 2008. In study 1, patients with K. pneumoniae BSIs were allocated among 3 groups according to isolate susceptibility profile: (1) carbapenem‐susceptible insolates (control group), (2) MBL‐producing isolates, or (3) KPC‐producing isolates. The MBL and KPC groups were compared with the control group to identify risk factors for development of K. pneumoniae BSI. In study 2, patients with K. pneumoniae BSIs who died were compared with survivors to identify risk factors for mortality. Results. Fifty‐nine patients had K. pneumoniae BSI...

Control of a multi-hospital outbreak of KPC-producing Klebsiella pneumoniae type 2 in France, September to October 2009.

Abstract: An outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae type 2 was detected in September 2009 in two hospitals in a suburb south of Paris, France. In total, 13 KPC-producing K. pneumoniae type 2 cases (four with infections and nine with digestive-tract colonisations) were identified, including a source case transferred from a Greek hospital. Of the 13 cases, seven were secondary cases associated with use of a contaminated duodenoscope used to examine the source case (attack rate: 41%) and five were secondary cases associated with patient-to-patient transmission in hospital. All isolated strains from the 13 patients: (i) exhibited resistance to all antibiotics except gentamicin and colistin, (ii) were more resistant to ertapenem (minimum inhibitory concentration (MIC) always greater than 4 mg/L) than to imipenem (MIC: 1-8 mg/L, depending on the isolate), (iii) carried the blaKPC-2 and blaSHV12 genes and (iv) had an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern. These cases occurred in three hospitals: some were transferred to four other hospitals. Extended infection control measures implemented in the seven hospitals included: (i) limiting transfer of cases and contact patients to other wards, (ii) cohorting separately cases and contact patients, (iii) reinforcing hand hygiene and contact precautions and (iv) systematic screening of contact patients. Overall, 341 contact patients were screened. A year after the outbreak, no additional case has been identified in these seven hospitals. This outbreak emphasises the importance of rapid identification and notification of emerging highly resistant K. pneumoniae strains in order to implement reinforced control measures. .

Prevalence of Antimicrobial-Resistant Pathogens in Canadian Hospitals: Results of the Canadian Ward Surveillance Study (CANWARD 2008)

Abstract: A total of 5,282 bacterial isolates obtained between 1 January and 31 December 31 2008, inclusive, from patients in 10 hospitals across Canada as part of the Canadian Ward Surveillance Study (CANWARD 2008) underwent susceptibility testing. The 10 most common organisms, representing 78.8% of all clinical specimens, were as follows: Escherichia coli (21.4%), methicillin-susceptible Staphylococcus aureus (MSSA; 13.9%), Streptococcus pneumoniae (10.3%), Pseudomonas aeruginosa (7.1%), Klebsiella pneumoniae (6.0%), coagulase-negative staphylococci/Staphylococcus epidermidis (5.4%), methicillin-resistant S. aureus (MRSA; 5.1%), Haemophilus influenzae (4.1%), Enterococcus spp. (3.3%), Enterobacter cloacae (2.2%). MRSA comprised 27.0% (272/1,007) of all S. aureus isolates (genotypically, 68.8% of MRSA were health care associated [HA-MRSA] and 27.6% were community associated [CA-MRSA]). Extended-spectrum β-lactamase (ESBL)-producing E. coli occurred in 4.9% of E. coli isolates. The CTX-M type was the predominant ESBL, with CTX-M-15 the most prevalent genotype. MRSA demonstrated no resistance to ceftobiprole, daptomycin, linezolid, telavancin, tigecycline, or vancomycin (0.4% intermediate intermediate resistance). E. coli demonstrated no resistance to ertapenem, meropenem, or tigecycline. Resistance rates with P. aeruginosa were as follows: colistin (polymyxin E), 0.8%; amikacin, 3.5%; cefepime, 7.2%; gentamicin, 12.3%; fluoroquinolones, 19.0 to 24.1%; meropenem, 5.6%; piperacillin-tazobactam, 8.0%. A multidrug-resistant (MDR) phenotype occurred frequently in P. aeruginosa (5.9%) but uncommonly in E. coli (1.2%) and K. pneumoniae (0.9%). In conclusion, E. coli, S. aureus (MSSA and MRSA), P. aeruginosa, S. pneumoniae, K. pneumoniae, H. influenzae, and Enterococcus spp. are the most common isolates recovered from clinical specimens in Canadian hospitals. The prevalence of MRSA was 27.0% (of which genotypically 27.6% were CA-MRSA), while ESBL-producing E. coli occurred in 4.9% of isolates. An MDR phenotype was common in P. aeruginosa.

In Vitro Activity of Fosfomycin against blaKPC-Containing Klebsiella pneumoniae Isolates, Including Those Nonsusceptible to Tigecycline and/or Colistin

Abstract: In vitro activity of fosfomycin was evaluated against 68 bla(KPC)-possessing Klebsiella pneumoniae (KpKPC) isolates, including 23 tigecycline- and/or colistin-nonsusceptible strains. By agar dilution, 93% of the overall KpKPC were susceptible (MIC(50/90) of 16/64 microg/ml, respectively). The subgroup of 23 tigecycline- and/or colistin-nonsusceptible strains showed susceptibility rates of 87% (MIC(50/90) of 32/128 microg/ml, respectively). Notably, 5 out of 6 extremely drug-resistant (tigecycline and colistin nonsusceptible) KpKPC were susceptible to fosfomycin. Compared to agar dilution, disk diffusion was more accurate than Etest.

Genetic Factors Associated with Elevated Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae

Abstract: In the United States, the most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is the production of a Klebsiella pneumoniae carbapenemase (KPC). KPC-producing isolates often exhibit a range of carbapenem MICs. To better understand the factors that contribute to overall carbapenem resistance, we analyzed 27 KPC-producing K. pneumoniae isolates with different levels of carbapenem resistance, 11 with low-level (i.e., meropenem or imipenem MIC ≤ 4 μg/ml), 2 with intermediate-level (i.e., meropenem and imipenem MIC = 8 μg/ml), and 14 with high-level (i.e., imipenem or meropenem MIC ≥ 16 μg/ml) carbapenem resistance, that were received from throughout the United States. Among 14 isolates that exhibited high-level carbapenem resistance, Western blot analysis indicated that 10 produced an elevated amount of KPC. These isolates either contained an increased bla(KPC) gene copy number (n = 3) or had deletions directly upstream of the bla(KPC) gene (n = 7). Four additional isolates lacked elevated KPC production but had high-level carbapenem resistance. Porin sequencing analysis identified 22 isolates potentially lacking a functional OmpK35 and three isolates potentially lacking a functional OmpK36. The highest carbapenem MICs were found in two isolates that lacked both functioning porins and produced elevated amounts of KPC. The 11 isolates with low-level carbapenem resistance contained neither an upstream deletion nor increased bla(KPC) copy number. These results suggest that both bla(KPC) copy number and deletions in the upstream genetic environment affect the level of KPC production and may contribute to high-level carbapenem resistance in KPC-producing K. pneumoniae, particularly when coupled with OmpK36 porin loss.

Transfer of carbapenem-resistant plasmid from Klebsiella pneumoniae ST258 to Escherichia coli in patient.

Abstract: Klebsiella pneumoniae carbapenemase (KPC) 3-producing Escherichia coli was isolated from a carrier of KPC-3-producing K. pneumoniae. The KPC-3 plasmid was identical in isolates of both species. The patient's gut flora contained a carbapenem-susceptible E. coli strain isogenic with the KPC-3-producing isolate, which suggests horizontal interspecies plasmid transfer.

Bacteremic community-acquired pneumonia due to Klebsiella pneumoniae: Clinical and microbiological characteristics in Taiwan, 2001-2008

Abstract: Klebsiella pneumoniae is the major cause of community-acquired pyogenic infections in Taiwan. This retrospective study evaluated the clinical and microbiological characteristics of bacteremic community-acquired pneumonia due to K. pneumoniae in Taiwanese adults. The clinical characteristics of bacteremic community-acquired pneumonia (CAP) in adults due to K. pneumoniae were compared to those of adults with bacteremic CAP due to Streptococcus pneumoniae at a tertiary medical center in Taiwan from 2001-2008. Risk factors for mortality of bacteremic CAP due to K. pneumoniae were analyzed. All clinical isolates of K. pneumoniae were examined for capsular serotypes, hypermucoviscosity phenotype, aerobactin and rmpA gene. K. pneumoniae was the dominant cause of bacteremic CAP and was associated with a more fulminant course and a worse prognosis than bacteremic CAP due to Streptococcus pneumoniae. Initial presentation with septic shock and respiratory failure were independent risk factors for both early and total mortality. Serotype K1 and K2 comprised around half of all isolates. There were no significant differences in the clinical characteristics of patients with bacteremic CAP due to K1/K2 and non-K1/K2 isolates. Hypermucoviscosity phenotype as well as the aerobactin and rmpA genes were highly prevalent in the K. pneumoniae isolates. K. pneumoniae continued to be the dominant cause of bacteremic CAP in Taiwanese adults during 2001-2008. Initial presentation with septic shock and respiratory failure were independent risk factors for both early and total mortality from K. pneumoniae bacteremic CAP. Serotypes K1/K2 comprised around half of all isolates, but did not predispose patients to a poor clinical outcome. Physicians should be aware of the poor prognosis of any patient with bacteremic K. pneumoniae CAP and monitor these patients more closely.

Complete Nucleotide Sequence of Klebsiella pneumoniae Multidrug Resistance Plasmid pKP048, Carrying blaKPC-2, blaDHA-1, qnrB4, and armA

Abstract: The Klebsiella pneumoniae multidrug resistance plasmid pKP048 was completely sequenced. This plasmid carries several important resistance determinants, such as bla(KPC-2), bla(DHA-1), qnrB4, and armA, which confer resistance to carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, respectively. Analysis of the finished 151,188-bp sequence data revealed 163 putative genes, 108 of which were assigned functions such as replication, stable inheritance, antibiotic resistance, a mobile element, conjugal transfer, and a restriction-modification system, showing the strong phylogenetic mosaicism and plasticity of the plasmid.

In Vitro Evaluation of Antibiotic Synergy for Polymyxin B-Resistant Carbapenemase-Producing Klebsiella pneumoniae

Abstract: Since carbapenemase-producing Klebsiella pneumoniae strains were first reported in North Carolina, these highly resistant organisms have been isolated with increasing frequency, especially in the New York City area. Polymyxin B is one of the few antimicrobials that retain reliable activity against these organisms. However, polymyxin B MICs are elevated against K. pneumoniae isolates with increasing frequency, leaving clinicians with few therapeutic options. We investigated several antimicrobial agents for potential synergy with polymyxin B against 12 clinical strains of carbapenemase-producing K. pneumoniae. A broth microdilution assay using a 96-well plate was developed in which graded dilutions of polymyxin B and the study drug were incubated with resistant isolates in a checkerboard pattern. Polymyxin B was studied in combination with cefazolin, ceftriaxone, cefepime, imipenem, gentamicin, tigecycline, doxycycline, and rifampin. All K. pneumoniae strains tested positive for K. pneumoniae carbapenemase (KPC) genes by real-time PCR and had elevated polymyxin B MIC values ranging from 16 to 128 μg/ml. Synergy was observed with the combination of polymyxin B and rifampin as well as with polymyxin B and doxycycline, resulting in at least a 4-fold decrease in the polymyxin B MIC. For both combinations, this effect occurred at physiologically achievable concentrations. Less pronounced synergy was noted with tigecycline and polymyxin B. No synergy was observed at physiologic concentrations with the other antimicrobials studied. These results suggest that rifampin, doxycycline, and tigecycline may be useful additions to polymyxin B in the treatment of infections caused by highly resistant carbapenemase-producing K. pneumoniae. Further studies are warranted to determine if these in vitro findings translate into clinical efficacy.

An Ertapenem-Resistant Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae Clone Carries a Novel OmpK36 Porin Variant

Abstract: Carbapenem-resistant Klebsiella pneumoniae caused an outbreak in a hospital in Rome, Italy. The clinical isolates were tested by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, multilocus sequence typing, plasmid typing, and β-lactamase identification. The OmpK35 and OmpK36 porins were analyzed by SDS-PAGE, and their genes were amplified and sequenced. Complementation experiments were performed using a recombinant unrelated ompK36 gene. An ertapenem-resistant and imipenem- and meropenem-susceptible clone was identified and assigned to the sequence type 37 lineage by MLST; it carried SHV-12 and CTX-M-15 ESBLs, did not produce the OmpK35 due to a nonsense mutation, and expressed a novel OmpK36 variant (OmpK36V). This variant showed two additional amino acids located within the L3 internal loop, one of the highly conserved domains of the protein. Two isolates of the same clone also exhibited resistance to imipenem and meropenem, due to the loss of OmpK36 expression by a nonsense mutation occurring in the ompK36V variant gene. These were the first carbapenem-resistant K. pneumoniae isolates identified within the hospital. Screening for the ompK36V gene of unrelated K. pneumoniae isolates derived from patients from 2006 to 2009 demonstrated the high frequency of this gene variant as well as its association with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem.

Emergence of a colistin-resistant KPC-2-producing Klebsiella pneumoniae ST258 clone in Hungary.

Abstract: Nine Klebsiella pneumoniae isolates showing non-susceptibility to carbapenems were collected from three centres in the north-eastern region of Hungary. The minimum inhibitory concentrations (MICs) of antibiotics were determined by Etest. The putative production of a carbapenemase was tested by the modified Hodge test. The presence of blaKPC genes was verified by polymerase chain reaction (PCR) and sequencing. Furthermore, molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates showed extensively drug-resistant (XDR) phenotype, and of these, eight isolates were highly resistant to colistin. The isolates carried blaKPC-2, blaSHV-12, blaTEM-1 and blaSHV-11. PFGE analysis of the nine KPC-2-producing Hungarian ST258 K. pneumoniae isolates, two KPC-2-producing Norwegian ST258 isolates and 33 CTX-M-15-producing ST11 isolates revealed the existence of one genetic cluster at an 88% similarity level. The overall results of the PFGE clustering, MLST and the presence of SHV-11 in both ST11 and ST258 suggest that this is the first hyperepidemic clonal complex of multidrug-resistant K. pneumoniae, probably CC258/CC340, possibly undergoing worldwide spread.

In Vitro Double and Triple Bactericidal Activities of Doripenem, Polymyxin B, and Rifampin against Multidrug-Resistant Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli

Abstract: In vitro double and triple bactericidal activities of doripenem, polymyxin B, and rifampin were assessed against 20 carbapenem-resistant clinical isolates with different mechanisms of carbapenem resistance. Bactericidal activity was achieved in 90% of all bacteria assayed using combinations of polymyxin B, doripenem, and rifampin against five each of the carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli isolates studied. Combinations with these antibacterials may provide a strategy for treatment of patients infected with such organisms.

Complete Nucleotide Sequence of KPC-3-Encoding Plasmid pKpQIL in the Epidemic Klebsiella pneumoniae Sequence Type 258

Abstract: We have determined the entire DNA sequence of plasmid pKpQIL, the blaKPC-3-carrying plasmid harbored by the carbapenem-resistant Klebsiella pneumoniae clone sequence type 258 (ST 258) in Israel. pKpQIL is a 113,637-bp, self-transmissible plasmid that belongs to the incompatibility group IncFII. It consists of a large backbone of a pKPN4-like plasmid and carries the blaKPC-3-containing Tn4401a transposon of a pNYC-like plasmid. Bacterial plasmids are extrachromosomal genetic elements that play a crucial role in the acquisition and dissemination of antibiotic resistance determinants through inter- and intraspecies horizontal gene transfer. Resistance to carbapenems in Enterobacteriaceae does not occur naturally but rather arises by the acquisition of various -lactamases encoded by transferrable plasmids (15). During the last decade, carbapenem-resistant KPC-producing Enterobacteriaceae strains, particularly Klebsiella pneumoniae, have emerged and spread globally (14). These KPCproducing strains harbor plasmids encoding KPC-type carbapenemases. Several KPC-encoding plasmids from K. pneumoniae were partially or fully sequenced, including blaKPC-2- and blaKPC-3-carrying plasmids from the United States (6) and blaKPC-2-carrying plasmids from Greece, Colombia (12), and China (19). These plasmids differed in size and harbored the transposon Tn4401, proven to be involved with the dissemination of blaKPC (12, 14). This element has been found entirely or in part in all of the blaKPC-encoding plasmid sequences to date and has been found in one of two isoforms, a or b, that differ by a 100-bp deletion upstream of the blaKPC gene (6). A variant of this transposon, harboring ISKpn8, was reported from China (19). In 2006, an extremely drug-resistant KPC-3-producing K. pneumoniae strain emerged in Israel (8), causing a nationwide clonal outbreak. This strain is genetically related to K. pneumoniae outbreak isolates from various regions in the United States (13), identified as sequence type 258 (ST 258) (7). Soon thereafter, this sequence type was found to have spread further geographically to China and several countries within the Middle East, Europe, and South America (1, 4, 11, 20). The K. pneumoniae ST 258 strain isolated in Israel is susceptible only to gentamicin and colistin (8), thereby limiting therapeutic options and thus posing a clinical and public health threat (18). Clinical isolates of K. pneumoniae ST 258 studied during the last 4 years in Israel have been found to harbor an identical blaKPC-3-encoding plasmid of about 105 kb (13), designated pKpQIL (9). This plasmid was shown to be self-conjugative and exclusively responsible for carbapenem resistance in these isolates. pKpQIL contains the Tn4401 element and TEM-1 gene (9). In this paper, we report the complete sequence and analysis of pKpQIL. Plasmid pKpQIL was purified from a representative clinical isolate of carbapenem-resistant KPC-3-producing K. pneumoniae ST 258, isolate 557. This isolate harbored two plasmids: pKpQIL and an additional 240-kb plasmid (9). Plasmid DNA was purified using a NucleoBond PC 100 plasmid midi kit

Risk Factors and Outcomes Associated with Acquisition of Colistin-Resistant KPC-Producing Klebsiella pneumoniae: a Matched Case-Control Study

Abstract: A matched 1:3 case-control study investigated factors predicting colistin-resistant versus colistin-susceptible KPC-producing Klebsiella pneumoniae acquisition and its impact on patient outcomes. Case patients were more often admitted from other institutions (P = 0.019) and had longer therapy with β-lactam/β-lactamase inhibitors (P = 0.002) and higher overall mortality (P = 0.05). All 52 study isolates were clonally related, suggesting horizontal dissemination. None of these parameters independently predicted colistin resistance, which probably occurred in a susceptible KPC-KP strain that was subsequently disseminated horizontally.

Klebsiella pneumoniae Capsule Polysaccharide Impedes the Expression of β-Defensins by Airway Epithelial Cells

Abstract: Human β-defensins (hBDs) contribute to the protection of the respiratory tract against pathogens. It is reasonable to postulate that pathogens have developed countermeasures to resist them. Klebsiella pneumoniae capsule polysaccharide (CPS), but not the lipopolysaccharide O antigen, mediated resistance against hBD1 and hBD2. hBD3 was the most potent hBD against Klebsiella. We investigated the possibility that as a strategy for survival in the lung, K. pneumoniae may not activate the expression of hBDs. Infection of A549 and normal human bronchial cells with 52145-ΔwcaK2, a CPS mutant, increased the expression of hBD2 and hBD3. Neither the wild type nor the lipopolysaccharide O antigen mutant increased the expression of hBDs. In vivo, 52145-ΔwcaK2 induced higher levels of mBD4 and mBD14, possible mouse orthologues of hBD2 and hBD3, respectively, than the wild type. 52145-ΔwcaK2-dependent upregulation of hBD2 occurred via NF-κB and mitogen-activated protein kinases (MAPKs) p44/42, Jun N-terminal protein kinase (JNK)-dependent pathways. The increase in hBD3 expression was dependent on the MAPK JNK. 52145-ΔwcaK2 engaged Toll-like receptors 2 and 4 (TLR2 and TLR4) to activate hBD2, whereas hBD3 expression was dependent on NOD1. K. pneumoniae induced the expression of CYLD and MKP-1, which act as negative regulators for 52145-ΔwcaK2-induced expression of hBDs. Bacterial engagement of pattern recognition receptors induced CYLD and MKP-1, which may initiate the attenuation of proinflammatory pathways. The results of this study indicate that K. pneumoniae CPS not only protects the pathogen from the bactericidal action of defensins but also impedes their expression. These features of K. pneumoniae CPS may facilitate pathogen survival in the hostile environment of the lung.

A survey on urinary tract infections associated with the three most common uropathogenic bacteria.

Abstract: Objectives: The most common uropathogenic Gram negative bacteria are Escherichia coli and Klebsiella pneumoniae. The purpose of this study was to determine the three most frequent bacterial agents causing Urinary Tract Infections (UTI) in patients who referred to Central Laboratory of Dr. Shariati Hospital, during 2 years (January 2006- December 2007). Materials and methods: The registered data were checked and collected by the questionnaires as a retrospective epidemiological survey. Then, the Chi Square tests were performed by SPSS software. Outcomes: The results of the present survey revealed that, the first two bacterial agents in different seasons were similar through the surveillance while the third one was variable. The first two frequent bacteria were respectively, Escherichia coli and Klebsiella pneumoniae. Conclusion: The gram negative bacteria of Escherichia coli and Klebsiella pneumoniae were the most common uropathogenic bacteria causing UTI. According to the statistical calculations, there was significant association between UTI caused by Escherichia coli and female gender (p<0.05).

Emergence of KPC-producing Pseudomonas aeruginosa in the United States

Abstract: Recent years have shown the emergence and dissemination of isolates of Enterobacteriaceae producing carbapenemases in different parts of the world (3). In many cases, those carbapenemases were KPC -lactamases (5). Those enzymes hydrolyze all -lactams, including carbapenems at a significant level, with the exception of cephamycins. The blaKPC-like genes have been reported most often from enterobacterial species (mostly Klebsiella pneumoniae species) recovered from several states in the United States (5). Besides the United States, KPC-producing K. pneumoniae isolates are found to be endemic in Greece and Israel, and there are, in addition, scattered reports from all over the world, including Western Europe, China, and South and Central America (5). In Colombia, the first identification of KPC-2-positive Pseudomonas aeruginosa isolates has been reported (6). We describe here the first identification of a KPC-producing P. aeruginosa isolate now in the United States. In October 2009, a 68-year-old African-American man with history of diabetes and hypertension was admitted with a myocardial infarction to a 1,500-bed teaching hospital in south Florida. Mechanical ventilation was required upon admission to the medical intensive care unit. He did not report any recent history of hospitalization or travel. The patient received an empirical antibiotic therapy consisting of ceftriaxone and vancomycin. Four weeks after admission, he developed hypothermia and blood and urine cultures grew P. aeruginosa. The patient subsequently received an empirical therapy based on meropenem. MICs of the P. aeruginosa P13 isolate measured by the Etest method (AB Biodisk, Solna, Sweden) and interpreted according to CLSI standards showed multidrug resistance including resistance to all carbapenems (carbapenem MICs of 256 g/ml) (2). That isolate remained susceptible only to amikacin, gentamicin, and colistin. Consequently, the therapy was based on colistin and amikacin, and his subsequent blood cultures remained negative. Molecular investigations were then performed on this isolate. PCR primers were used for the detection of Ambler class A and class B -lactamase genes, followed by sequencing, which identified the blaKPC-2 -lactamase gene coding for carbapenemase KPC-2 (8). Analysis of the plasmid content of P. aeruginosa isolate 13 identified a single plasmid of ca. 66 kb that was successfully transferred to Escherichia coli by electroporation, with a selection performed on amoxicillin (100 g/ml)containing agar plates. The E. coli transformants expressing KPC-2 showed a 3-fold increase of MICs for imipenem, meropenem, and ertapenem, but they did not show any additional non--lactam resistance. PCR mapping performed as described previously (3) showed that the blaKPC-2 gene was part of the Tn4401b transposon originally identified from a K. pneumoniae isolate from New York (4) and also identified from the clonally related Colombian P. aeruginosa isolates (6). Pulsed-field gel electrophoresis (PFGE) performed as described previously, however, indicated that P. aeruginosa isolate P13 was clonally unrelated to P. aeruginosa PA2404 from Colombia (data not shown). This is the first identification of a KPC-producing P. aeruginosa isolate in the United States. It is noteworthy that it did not correspond to an imported case. It therefore remains to be evaluated to what extent KPC-type enzymes have spread in P. aeruginosa in the United States, since the phenotypic detection of production of that carbapenemase remains impossible. Use

Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India.

Abstract: Background & objectives Extended spectrum beta lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. The enzymes are predominantly plasmid mediated and are derived from broad-spectrum beta lactamase TEM-1, TEM-2 or SHV-1 by a limited number of mutations. This study was undertaken to characterize ESBL producers among Escherichia coli and Klebsiella pneumoniae by PCR-RFLP, which were initially screened by phenotypic method. Methods A total of 100 isolates of each species (E. coli and K. pneumoniae) were screened for ESBL production. PCR analysis for β-lactamase genes of the family TEM and SHV was also carried out. PCR products of TEM and SHV genes were subjected to digest with three different restriction enzymes. The digested products were run on 1.5 per cent agarose gel, stained and examined for DNA bands. Results PCR carried out on plasmid DNA alone detected 30 per cent ESBL positive isolates using TEM primer and 38 per cent using SHV primer, whereas PCR for both plasmid and chromosomal DNA showed 56 per cent positivity for TEM and 60 per cent positivity for SHV. Interpretation & conclusion RFLP yielded homogeneous band pattern, suggesting that there may be a point source or a common evolutionary origin for all the ESBL isolates.

ramR Mutations in Clinical Isolates of Klebsiella pneumoniae with Reduced Susceptibility to Tigecycline

Abstract: Five Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline (MIC, 2 microg/ml) were analyzed. A gene homologous to ramR of Salmonella enterica was identified in Klebsiella pneumoniae. Sequencing of ramR in the nonsusceptible Klebsiella strains revealed deletions, insertions, and point mutations. Transformation of mutants with wild-type ramR genes, but not with mutant ramR genes, restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. Thus, this study reveals a molecular mechanism for tigecycline resistance in Klebsiella pneumoniae.