Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13.

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling.

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC ß-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism of resistance of K. pneumoniae during carbapenem treatment. RESULTS: Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 was found to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored blaSHV-187 and blaTEM-116. The blaSHV-187 and blaTEM-116 genes were not detected in FK-2723 and FK-2820. blaDHA-1, qnrB4, aac (6')-IIc, and blaSHV-12, EreA2, CatA2, SulI, and tetD, were identified in both FK-2723 and FK-2820. Moreover, the genes blaDHA-1, qnrB4, aac (6')-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 non-sense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate. CONCLUSION: This study in China highlight that the alteration of outer membrane porins due to the 14-day use of imipenem play a potential role in leading to clinical presentation of carbapenem-resistance. This is the first description of increased resistance developing from a carbapenem-susceptible K. pneumoniae with imipenem treatment driven by outer membrane remodeling.

Successful control of the first carbapenem-resistant Klebsiella pneumoniae outbreak in a Chinese hospital 2017-2019.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is considered as a serious global threat. CRKPs occurred only sporadically in the Second Hospital of Shanxi Medical University. Our study aimed to investigate and control the first outbreak of CRKP in our hospital occurred between October 2017 and August 2019. METHODS: The antimicrobial stewardship (AMS) workers have been implemented control measures properly. Clinical and epidemiological data were retrospectively collected from medical records. Carbapenemase genes were detected by modified carbapenem inactivation method (mCIM) test and the EDTA-modified carbapenem inactivation method (eCIM) test. Resistance genes were identified by polymerase chain reaction (PCR) and sequencing. Genetic relatedness was studied by multilocus sequence typing (MLST). RESULTS: During the outbreak, 31 patients were infected with CRKP isolates. 20 (64.5%) patients were infected with KPC-2 and/or NDM-1 producing K. pneumoniae. Mostly MLST-sequence types belonged to ST11 (21/31). The outbreak was two major K. pneumoniae clusters present in epidemiologically linked patients. CONCLUSIONS: Setting up AMS workers is potentially a highly efficient strategy for the successful control of the outbreak. A multimodal and multidisciplinary infection control strategy proved to be crucial. The emergence of CRKP in our hospital emphasizes the importance of continuous monitoring of these isolates, which helps to limit the spread of CRKPs and improve the level of management.

Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania.

We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.

Bacterial isolation and antibiotic susceptibility from diabetic foot ulcers in Kenya using microbiological tests and comparison with RT-PCR in detection of S. aureus and MRSA.

OBJECTIVES: Diabetic foot ulcers (DFUs) often lead to hospital admissions, amputations and deaths; however, there is no up-to-date information on microbial isolates from DFUs and no mention of utilization of molecular techniques in Sub-Saharan Africa. We conducted a cross-sectional study among 83 adult patients at a tertiary hospital in Kenya over 12 months. The study aimed to isolate, identify bacteria, their antibiotic susceptibility patterns in active DFUs, and to compare standard microbiological methods versus a real-time PCR commercial kit in the detection of Staphylococcus aureus DNA and methicillin-resistant S. aureus (MRSA) DNA. RESULTS: Eighty swabs (94%) were culture-positive; 29% were Gram-positive and 65% were Gram-negative. The main organisms isolated were S. aureus (16%), Escherichia coli (15%), Proteus mirabilis (11%), Klebsiella pneumoniae (7%) and Pseudomonas aeruginosa (7%). The bacterial isolates showed resistance to commonly used antibiotics such as ampicillin, amoxicillin, cefepime, ceftazidime, cefuroxime, clindamycin, erythromycin, piperacillin-tazobactam, tetracycline and trimethoprim-sulphamethoxazole (TMPSMX). Thirty-one percent of the S. aureus isolated and 40% of the Gram-negatives were multi-drug resistant organisms (MDROs). There was a high prevalence of nosocomial bacteria. MRSA were not identified using culture methods but were identified using PCR. PCR was more sensitive but less specific than culture-based methods to identify S. aureus.

RS sample: Can be guide for empirical treatment of haematological malignancy patients?

Neutropenia due to intensive chemotherapy in haematological malignancy patients leaves the host vulnerable and makes them susceptible to infections. Infections are the most important cause of morbidity and mortality especially in haematological malignancy and chemotherapy patients. In addition, the use of multiple or inappropriate antibiotics leads to the development of resistant microorganisms. Therefore, the choice of empirical treatment is of vital important in these patient groups. Escherichia coli, Acinetobacter baumannii and Klebsiella pneumoniae are among the most frequently isolated Gram negative bacteria in neutropenic patients. Rectal swab (RS) samples were obtained from haematological malignancy patients not yet on chemotherapy or have no infection on chemotherapy period, E. coli was isolated from these samples, and A. baumannii and K. pneumoniae colonization were investigated. Susceptibilities of bacteria against antibiotics used in empirical treatment and prophylaxis were determined by using Gradient test strips according to the EUCAST recommendation. All isolates were sensitive against colistin. The resistant rates of antibiotics were detected as 39.1%, 9.4%, 6.8%, 35.1%, 31%, 39.1% for ciprofloxacin, meropenem, imipenem, piperacillin-tazobactam, cefepime, ceftazidime respectively The clonal relationship between Gram negative bacteria of intestinal flora and infection agents of same patient was investigated by Pulsed-Field gel electrophoresis. Twenty-three of the 30 patients (76.6%) were found to have a clonal relationship between the bacterial isolates before and after infection. It was determined that it can be able to predict with RS samples about possible agents of infection and their antibiotic susceptibility patterns.

Intra-hospital differences in antibiotic use correlate with antimicrobial resistance rate in Escherichia coli and Klebsiella pneumoniae: a retrospective observational study.

Background: Monitoring antimicrobial use and resistance in hospitals are important tools of antimicrobial stewardship programs. We aimed to determine the association between the use of frequently prescribed antibiotics and the corresponding resistance rates in Escherichia coli and Klebsiella pneumoniae among the clinical departments of a tertiary care hospital. Methods: We performed a retrospective observational study to analyse the use of nine frequently prescribed antibiotics and the corresponding antimicrobial resistance rates in hospital acquired E. coli and K. pneumoniae isolates from 18 departments of our institution over 9 years (2008-2016). The main cross-sectional analysis assessed the hypothetical influence of antibiotic consumption on resistance by mixed logistic regression models. Results: We found an association between antibiotic use and resistance rates in E. coli for amoxicillin-clavulanic acid (OR per each step of 5 defined daily dose/100 bed-days 1.07, 95% CI 1.02-1.12; p = 0.004), piperacillin-tazobactam (OR 2.11, 95% CI 1.45-3.07; p < 0.001), quinolones (OR 1.52, 95% CI 1.25-1.86; p < 0.001) and trimethoprim-sulfamethoxazole (OR 1.59, 95% CI 1.19-2.13; p = 0.002). Additionally, we found a significant association when all nine antibiotics were combined in one analysis. The association between consumption and resistance rates was stronger for nosocomial than for community strains. In K. pneumoniae, we found an association for amoxicillin-clavulanic acid (OR 1.07, 95% CI 1.01-1.14; p = 0.025) and for trimethoprim-sulfamethoxazole (OR 2.02, 95% CI 1.44-2.84; p < 0.001). The combined analysis did not show an association between consumption and resistance (OR 1.06, 95% CI 0.99-1.14; p = 0.07). Conclusions: We documented an association between antibiotic use and resistance rate for amoxicillin-clavulanic acid, piperacillin-tazobactam, quinolones and trimethoprim-sulfamethoxazole in E. coli and for amoxicillin-clavulanic acid and trimethoprim-sulfamethoxazole in K. pneumoniae across different hospital departments. Our data will support stewardship interventions to optimize antibiotic prescribing at a department level.

Ação dos extratos de hamamélis e abacateiro sobre cepas clínicas resistentes de Klebsiella pneumoniae e Pseudomonas aeruginosa / Action of Hamamelis and Avocado extracts on clinical strains of resistant Klebsiella pneumoniae and Pseudomonas aeruginosa

A resistência adquirida pelas bactérias aos antibióticos de amplo espectro tornou-se uma ameaça à saúde global. A necessidade de encontrar fármacos que consigam combater micro-organismos multirresistentes tem se tornado um grande desafio. Neste cenário, as plantas medicinais são promissoras por terem propriedades antimicrobianas eficazes, devido à presença de compostos fitoquímicos com atividades biológicas diversas. O objetivo desse estudo foi avaliar a ação antimicrobiana dos extratos glicólicos de Hamamelis virginiana (hamamélis) e de Persea americana (abacateiro) sobre cepas multirresistentes de Klebsiella pneumoniae e Pseudomonas aeruginosa. A avaliação da atividade antimicrobiana dos extratos vegetais foi realizada em sete cepas clínicas de K. pneumoniae e de P. aeruginosa, em comparação com uma cepa de referência de K. pneumoniae (ATCC 4352) e de P. aeruginosa (ATCC 15442). Para a determinação das concentrações inibitórias mínimas (CIM) e bactericida mínima (CBM) dos extratos de abacateiro e de hamamélis foi utilizado o método de microdiluição em caldo, segundo NCCLS. Após obtenção destes resultados, foi verificada a ação dos extratos sobre biofilmes monomicrobianos de oito cepas de K. pneumoniae e de P. aeruginosa (1 cepa ATCC e 7 cepas clínicas resistentes). Após o período de 48 horas para a formação do biofilme, os extratos foram adicionados separadamente, pelo período de cinco minutos, na concentração efetiva pré-determinada (CBM) e concentrações superiores. Posteriormente, os biofilmes foram lavados e mensurados por dois diferentes testes, em que foram avaliadas a biomassa do biofilme pelo cristal violeta e a viabilidade dos micro-organismos pelo teste de MTT. Os experimentos foram realizados com n=10, utilizando duas repetições para cada cepa/extrato. Os dados foram analisados estatisticamente pelo método ANOVA, complementado pelo Teste de Tukey, com nível de significância de 5% (p≤0.05). Os resultados demonstraram uma significativa atividade antibacteriana de ambos os extratos contra a cepapadrão e as cepas clínicas multirresistentes de K. pneumoniae e P. aeruginosa. Os extratos de abacateiro e de hamamélis, em todas as concentrações, demonstraram uma redução estatisticamente significativa na biomassa formada para pelo menos alguma das cepas clínicas de K. pneumoniae e P. aeruginosa testada. Para avaliação da viabilidade celular, ambos os extratos, na concentração de 200 mg/mL, demonstraram redução estatisticamente significativa nas cepas multirresistentes avaliadas de K. pneumoniae e P. aeruginosa. Como conclusão, os extratos glicólicos de H. virginiana e P. americana apresentaram ação antimicrobiana, resultando em concentrações com capacidade bactericida e significativa redução antibiofilme formados pelas cepas multirresistentes e ATCC de K. pneumoniae e P. aeruginosa(AU)

The resistance created by bacteria to broad-spectrum antibiotics has become a threat to global health. The need to find drugs that can combat multidrug-resistant microorganisms has become a major challenge. In this scenario, medicinal plants are promising because they have effective antimicrobial properties due to the presence of phytochemical compounds with diverse biological activities. The objective of this study was to evaluate the antimicrobial action of the glycolic extracts of Hamamelis virginiana (hamamelis) and Persea americana (avocado) on multiresistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa. The evaluation of the antimicrobial activity of the plant extracts was carried out in seven clinical strains of K. pneumoniae and P. aeruginosa, compared to an strain of K. pneumoniae (ATCC 4352) and P. aeruginosa (ATCC 15442). The broth microdilution method, according to NCCLS, was used to determine the minimum inhibitory (MIC) and bactericidal (MBM) concentrations of the extracts of avocado and witch hazel. After obtaining these results, the extracts on monomicrobial biofilms of eight strains of K. pneumoniae and P. aeruginosa (1 strain ATCC and 7 resistant clinical strains) were verified. After the 48 hour period for biofilm formation, the extracts were removed, with a five-minute interval, in the effective exercise session (MBC) and in the upper sets. Afterwards, the biofilms were washed and measured by two different testicles, in which the biofilm biomass was evaluated by the violet glass and the viability of the microorganisms by the MTT test. The experiments were performed with n = 10, using two replicates for each strain / extract. The data were analyzed statistically by the ANOVA method, complemented by the Tukey test, with a significance level of 5% (p≤0.05). The results demonstrated an antibacterial activity of both extracts against the standard strain and as the clinical strains of K. pneumoniae and P. aeruginosa. The extracts of avocado and witch hazel at all concentrations demonstrated a statistically significant reduction in the biomass formed for at least some of the clinical strains of K. pneumoniae and P. aeruginosa tested. For evaluation of cell viability, both extracts, at a concentration of 200 mg / mL, demonstrated a statistically significant reduction in the multiresistant strains evaluated for K. pneumoniae and P. aeruginosa. In conclusion, the glycolic extracts of H. virginiana and P. americana showed antimicrobial action, resulting in concentrations with bactericidal capacity and significant antibiofilm reduction formed by the multiresistant strains and ATCC of K. pneumoniae and P. aeruginosa(AU)

Colistin Resistance in Carbapenem-Resistant Klebsiella pneumoniae: Laboratory Detection and Impact on Mortality.

Background: Polymyxins including colistin are an important "last-line" treatment for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKp). Increasing use of colistin has led to resistance to this cationic antimicrobial peptide. Methods: A cohort nested within the Consortium on Resistance against Carbapenems in Klebsiella pneumoniae (CRACKLE) was constructed of patients with infection, or colonization with CRKp isolates tested for colistin susceptibility during the study period of December, 2011 to October, 2014. Reference colistin resistance determination as performed by broth macrodilution was compared to results from clinical microbiology laboratories (Etest) and to polymyxin resistance testing. Each patient was included once, at the time of their first colistin-tested CRKp positive culture. Time to 30-day in-hospital all-cause mortality was evaluated by Kaplan-Meier curves and Cox proportional hazard modeling. Results: In 246 patients with CRKp, 13% possessed ColR CRKp. ColR was underestimated by Etest (very major error rate = 35%, major error rate = 0.4%). A variety of rep-PCR strain types were encountered in both the ColS and the ColR groups. Carbapenem resistance was mediated primarily by blaKPC-2 (46%) and blaKPC-3 (50%). ColR was associated with increased hazard for in-hospital mortality (aHR 3.48; 95% confidence interval, 1.73-6.57; P < .001). The plasmid-associated ColR genes, mcr-1 and mcr-2 were not detected in any of the ColR CRKp. Conclusions: In this cohort, 13% of patients with CRKp presented with ColR CRKp. The apparent polyclonal nature of the isolates suggests de novo emergence of ColR in this cohort as the primary factor driving ColR. Importantly, mortality was increased in patients with ColR isolates.

Clonal dissemination of multilocus sequence type ST15 KPC-2-producing Klebsiella pneumoniae in Bulgaria.

A total of 36 consecutive clinical and two fecal-screening carbapenem-resistant Klebsiella pneumoniae isolates from two Bulgarian university hospitals (Varna and Pleven) were investigated. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and multilocus sequence typing (MLST). Most of the isolates demonstrated multidrug resistance profile. Amikacin and tigecycline retained good activity with susceptibility rates of 95 and 87%, respectively. The resistance rate to colistin was 63%. Six RAPD- and MLST-types were identified: the dominating MLST-type was ST15 (27 isolates), followed by ST76 (six isolates), and ST1350 (two isolates). ST101, ST258, and ST151 were detected once. All except one of the K. pneumoniae produced KPC-2, mostly in combination with CTX-M-15, while for one isolate (ST101) the enzymes OXA-48 and CTX-M-14 were found. All KPC-2-producing transconjugants revealed the presence of IncFII plasmid. The OXA-48- and CTX-M-14-producing isolate showed the presence of L/M replicon type. The dissemination of KPC-2-producing K.pneumoniae in Bulgaria is mainly due to the sustained spread of successful ST15 clone and to a lesser extent of ST76 clone. This is the first report of OXA-48 producing ST101 K. pneumoniae in Bulgaria.

Genomic epidemiology of Klebsiella pneumoniae in Italy and novel insights into the origin and global evolution of its resistance to carbapenem antibiotics.

Klebsiella pneumoniae is at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated with K. pneumoniae infections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniae strains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology of K. pneumoniae in Italy. Thirty-one strains were carbapenem-resistant K. pneumoniae carbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity of K. pneumoniae worldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼ 1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group of K. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding of K. pneumoniae evolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

Characterisation of the first ongoing outbreak due to KPC-3-producing Klebsiella pneumoniae (ST512) in Spain.

The aim of this study was to characterise carbapenem-resistant Klebsiella pneumoniae isolates that caused an outbreak in a hospital in the south of Spain, originating from a patient transferred in 2012 from Italy. Forty-four K. pneumoniae isolates, recovered from 28 patients, were screened by PCR for extended-spectrum ß-lactamase (ESBL) and carbapenemase genes and the products were further sequenced. Plasmids were transferred by electroporation and were classified using PCR-based Inc/rep typing and IncF subtyping. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to determine the genetic relatedness of the isolates. All isolates yielded positive modified Hodge test results, harboured bla(SHV-11), bla(TEM-1) and bla(KPC-3) genes, showed an identical PFGE pattern, and were assigned to clone sequence type 512 (ST512). The bla(KPC-3) gene was located on a 140-kb K2:A-:B-plasmid. In conclusion, the successful K. pneumoniae ST512 clone caused a major outbreak in Spain from an imported case and is the first description of an outbreak in this country due to the KPC-3-producing K. pneumoniae ST512 clone.

[Current data and trends about the resistance of Gram-negative pathogens to beta-lactams]. / Aktuelle Daten und Trends zur ß-Lactam-Resistenz bei gramnegativen Infektionserregern.

In recent years the resistance of Gram-negative pathogens to beta-lactam antibiotics, such as cephalosporins and carbapenems has increased. The resistant strains produce different beta-lactam hydrolysing enzymes (beta-lactamases). In particular extended spectrum beta-lactamases (ESBL) are prevalent in Escherichia coli and Klebsiella pneumoniae. The ESBL genes are located on different plasmids facilitating the transfer of resistance within a species and between different Gram-negative species. Within the scope of various studies the Robert Koch Institute in Wernigerode investigated ESBL-producing human Enterobacteriaceae using molecular methods. The results showed that distinct ESBL types, such as the CTX-M enzymes are dominant in Germany whereby CTX-M-15 and CTX-M-1 are the most prevalent variants in E. coli and K. pneumoniae. The aim of ongoing investigations within the RESET network project is to investigate the dissemination pathways of ESBL-producing bacteria in different settings (e.g. in humans, animals and food).

Rapid acquisition of decreased carbapenem susceptibility in a strain of Klebsiella pneumoniae arising during meropenem therapy.

A strain of Klebsiella pneumoniae (K1) was isolated from a catheterized patient with a urinary tract infection. The patient was subsequently treated with meropenem, after which K. pneumoniae (K2) was once again isolated from the patient's urine. Susceptibility testing showed that strain K1 was fully susceptible to carbapenem antibiotics with the exception of ertapenem, to which it exhibited intermediate resistance, whilst K2 was resistant to ertapenem and meropenem. From pulsed-field gel electrophoresis profiling both strains exhibited identical banding patterns. Both contained CTX-M-15, OXA-1, SHV-1 and TEM-1 ß-lactamase genes following PCR analyses. Outer membrane protein analysis demonstrated that K1 and K2 lacked an OMP of c. 40 kDa, with an additional OMP of c. 36 kDa missing from K2. Mutation studies showed that the K2 OMP phenotype could be selected by single-step carbapenem-resistant mutants of K1. Expression of transcriptional activator ramA and efflux pump component gene acrA were up-regulated in both strains by RT-PCR. Neither strain expressed ompK35, but ompK36 was found in both. Sequence analysis revealed gene sequences of ompK35, ompK36 and ramR in both strains; notably, ramR contained a mutation resulting in a premature stop codon. Transconjugation studies demonstrated transfer of a plasmid into E. coli encoding the CTX-M-15, TEM-1 and OXA-1 ß-lactamases. We concluded that the carbapenem-resistant phenotype observed from this patient was attributable to a combination of CTX-M-15 ß-lactamase, up-regulated efflux and altered outer membrane permeability, and that K2 arose from K1 as a direct result of meropenem therapy.

An outbreak of infection due to beta-Lactamase Klebsiella pneumoniae Carbapenemase 2-producing K. pneumoniae in a Greek University Hospital: molecular characterization, epidemiology, and outcomes.

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.

Epidemiology and risk factors for ESBL-producing Klebsiella pneumoniae: a case control study.

INTRODUCTION: Increased production of extended-spectrum ß-lactamases (ESBLs) has become an important issue for treatment of severe Klebsiella pneumoniae (K. pneumoniae) infections. This study aimed to evaluate risk factors of infection from ESBL-producing K. pneumoniae (ESBL-KP). METHODOLOGY: Risk factors were evaluated using a retrospective case control design. Fifty-two patients admitted to Firat University Hospital (FUH) with invasive infections from ESBL-KP were employed as cases. Patients admitted to FUH with non-ESBL-producing K. pneumoniae invasive infection were chosen as controls. Potential risk factors of the cases and controls were evaluated using hospital charts. Pulsed-field Gel Electrophoresis (PFGE) was used to show the relatedness of ESBL-KP strains. RESULTS: In univariate analysis, the following factors were found significant for ESBL-KP: pre-infection hospital stay, nosocomial origin, central venous catheterization, surgical intervention, antibiotic use longer than one week, and previous hospitalization. In contrast, stepwise logistic regression analysis showed that two variables, previous antibiotic use (p = 0.000) and surgical intervention (p = 0.006), remained significantly associated with risk for infection with an ESBL-KP. Molecular epidemiology identified several clusters among the ESBL-producing isolates. CONCLUSIONS: Antibiotic use and surgical intervention were significant associated factors for infections with ESBL-KP.

In vivo selection of OmpK35-deficient mutant after cefuroxime therapy for primary liver abscess caused by Klebsiella pneumoniae.

OBJECTIVES: The aim of the study was to characterize the genetic basis of beta-lactam resistance developed in clinical isolates of Klebsiella pneumoniae after exposure to cefuroxime. METHODS: Clinical features of two episodes of liver abscess caused by K. pneumoniae in a diabetic patient were reported. Four isolates (KP(1)/KP(2) and KP(3)/KP(4)) of K. pneumoniae were recovered from cultures of blood/pus in the first and second episodes, respectively. Laboratory investigation of the K. pneumoniae isolates included genotyping by PFGE, resistance gene analysis by PCR amplification and DNA sequencing, and outer membrane protein analysis by SDS-PAGE. RESULTS: KP(3) and KP(4) were recovered after a 21 day cefuroxime therapy and demonstrated identical genotypes to that of KP(1) and KP(2). However, compared with KP(1) and KP(2), emerging resistance to piperacillin, cefalotin, cefuroxime and cefoxitin was observed. The other antibiotics tested, except ampicillin, retained the same effectiveness against the four isolates, although increases (4- to 8-fold) in the MICs of cefotaxime, ceftriaxone, ceftazidime, cefepime, flomoxef and aztreonam were observed in KP(3) and KP(4). None of the isolates produced extended-spectrum beta-lactamases or plasmid-mediated AmpC beta-lactamases. Deficiency in the expression of an outer membrane protein (OmpK35) was observed in the cefuroxime-resistant isolates, KP(3) and KP(4). CONCLUSIONS: The increased resistance to cephalosporins in these clinical isolates of K. pneumoniae after exposure to cefuroxime might be related to the loss of OmpK35.

[Protective activity of a cell-free Klebsiella vaccine in relation to different Klebsiella pneumoniae serovars]. / Protektivnaia aktivnost' beskletochnoi klebsielleznoi vaktsiny v otnoshenii razlichnykh serovarov Klebsiella pneumoniae.

The capacity of dried Klebsiella cell-free vaccine, obtained from strain No. 204 by the disintegration of microbial cells with hydroxylamine, for protecting mice from Klebsiella septic infection caused by the homologous serovar and 9 heterologous serovars of K. pneumoniae was studied. The newly developed preparation was found capable of stimulating immunity not only to the homologous K. pneumoniae serovar, but also to other K. pneumoniae heterologous serovars: K1, K9, K11, K16, K20, K61. The protective capacity of the preparation with respect to these serovars was not inferior to that of the vaccines prepared by the same method from the corresponding homologous strains. The capacity of the vaccine to protect mice from Klebsiella sepsis was manifested irrespective of the virulence of the strains used for challenge.