Marek Gniadkowski

Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases.

Klebsiella pneumoniae carbapenemases (KPCs) were originally identified in the USA in 1996. Since then, these versatile β-lactamases have spread internationally among Gram-negative bacteria, especially K pneumoniae, although their precise epidemiology is diverse across countries and regions. The mortality described among patients infected with organisms positive for KPC is high, perhaps as a result of the limited antibiotic options remaining (often colistin, tigecycline, or aminoglycosides). Triple drug combinations using colistin, tigecycline, and imipenem have recently been associated with improved survival among patients with bacteraemia. In this Review, we summarise the epidemiology of KPCs across continents, and discuss issues around detection, present antibiotic options and those in development, treatment outcome and mortality, and infection control. In view of the limitations of present treatments and the paucity of new drugs in the pipeline, infection control must be our primary defence for now.

Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe

Plasmid-acquired carbapenemases in Enterobacteriaceae, which were first discovered in Europe in the 1990s, are now increasingly being identified at an alarming rate. Although their hydrolysis spectrum may vary, they hydrolyse most β-lactams, including carbapenems. They are mostly of the KPC, VIM, NDM and OXA-48 types. Their prevalence in Europe as reported in 2011 varies significantly from high (Greece and Italy) to low (Nordic countries). The types of carbapenemase vary among countries, partially depending on the cultural/population exchange relationship between the European countries and the possible reservoirs of each carbapenemase. Carbapenemase producers are mainly identified among Klebsiella pneumoniae and Escherichia coli, and still mostly in hospital settings and rarely in the community. Although important nosocomial outbreaks with carbapenemase-producing Enterobacteriaceae have been extensively reported, many new cases are still related to importation from a foreign country. Rapid identification of colonized or infected patients and screening of carriers is possible, and will probably be effective for prevention of a scenario of endemicity, as now reported for extended-spectrum β-lactamase (mainly CTX-M) producers in all European countries.

Interventions to reduce colonisation and transmission of antimicrobial-resistant bacteria in intensive care units: an interrupted time series study and cluster randomised trial.

Summary Background Intensive care units (ICUs) are high-risk areas for transmission of antimicrobial-resistant bacteria, but no controlled study has tested the effect of rapid screening and isolation of carriers on transmission in settings with best-standard precautions. We assessed interventions to reduce colonisation and transmission of antimicrobial-resistant bacteria in European ICUs. Methods We did this study in three phases at 13 ICUs. After a 6 month baseline period (phase 1), we did an interrupted time series study of universal chlorhexidine body-washing combined with hand hygiene improvement for 6 months (phase 2), followed by a 12–15 month cluster randomised trial (phase 3). ICUs were randomly assigned by computer generated randomisation schedule to either conventional screening (chromogenic screening for meticillin-resistant Staphylococcus aureus [MRSA] and vancomycin-resistant enterococci [VRE]) or rapid screening (PCR testing for MRSA and VRE and chromogenic screening for highly resistant Enterobacteriaceae [HRE]); with contact precautions for identified carriers. The primary outcome was acquisition of resistant bacteria per 100 patient-days at risk, for which we calculated step changes and changes in trends after the introduction of each intervention. We assessed acquisition by microbiological surveillance and analysed it with a multilevel Poisson segmented regression model. We compared screening groups with a likelihood ratio test that combined step changes and changes to trend. This study is registered with ClinicalTrials.gov, number NCT00976638. Findings Seven ICUs were assigned to rapid screening and six to conventional screening. Mean hand hygiene compliance improved from 52% in phase 1 to 69% in phase 2, and 77% in phase 3. Median proportions of patients receiving chlorhexidine body-washing increased from 0% to 100% at the start of phase 2. For trends in acquisition of antimicrobial-resistant bacteria, weekly incidence rate ratio (IRR) was 0·976 (0·954–0·999) for phase 2 and 1·015 (0·998–1·032) for phase 3. For step changes, weekly IRR was 0·955 (0·676–1·348) for phase 2 and 0·634 (0·349–1·153) for phase 3. The decrease in trend in phase 2 was largely caused by changes in acquisition of MRSA (weekly IRR 0·925, 95% CI 0·890–0·962). Acquisition was lower in the conventional screening group than in the rapid screening group, but did not differ significantly (p=0·06). Interpretation Improved hand hygiene plus unit-wide chlorhexidine body-washing reduced acquisition of antimicrobial-resistant bacteria, particularly MRSA. In the context of a sustained high level of compliance to hand hygiene and chlorhexidine bathings, screening and isolation of carriers do not reduce acquisition rates of multidrug-resistant bacteria, whether or not screening is done with rapid testing or conventional testing. Funding European Commission.

Identification and screening of carbapenemase-producing Enterobacteriaceae

Carbapenem-hydrolysing β-lactamases are the most powerful β-lactamases, being able to hydrolyse almost all β-lactams. They are mostly of the KPC, VIM, IMP, NDM and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern, as these carbapenemase producers are multidrug-resistant. Detection of infected patients and of carriers are the two main approaches for prevention of their spread. Phenotypic and molecular-based techniques are able to identify these carbapenemase producers, although with variable efficiencies. The detection of carriers still relies mostly on the use of screening culture media.

Carbapenemase-producing Enterobacteriaceae in Europe: A survey among national experts from 39 countries, February 2013

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a threat to healthcare delivery, although its extent differs substantially from country to country. In February 2013, national experts from 39 European countries were invited to self-assess the current epidemiological situation of CPE in their country. Information about national management of CPE was also reported. The results highlight the urgent need for a coordinated European effort on early diagnosis, active surveillance, and guidance on infection control measures.

Comparison of Multiple-Locus Variable-Number Tandem-Repeat Analysis with Pulsed-Field Gel Electrophoresis, spa Typing, and Multilocus Sequence Typing for Clonal Characterization of Staphylococcus aureus Isolates

ABSTRACT Multiple-locus variable-number tandem-repeat analysis (MLVA), a new PCR-based method of typing Staphylococcus aureus, was compared to pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) on a group of 59 S. aureus (mostly methicillin-resistant) clinical isolates. The aim of the study was to establish possible criteria of clustering MLVA patterns and to check concordance levels between the results produced by MLVA and the three other typing methods. As in our earlier study, MLVA turned out to have discriminatory power similar to that of PFGE. Comparison of data obtained by the two approaches allowed us to propose a 70% or ca. 80% cutoff value of the similarity between two MLVA patterns, depending on a cutoff level applied to interpret the PFGE results, 75% or ca. 90%, respectively. The cutoff values corresponded to the difference of up to six or four bands, respectively, among maximum 14 bands in total produced by two isolates in the analysis. The MLVA clusters matched well those obtained by PFGE, and they were also consistent in general with clusters generated by spa typing and MLST, these latter methods characterized lower resolution. Our results suggest that MLVA may be reliable in shorter-term S. aureus epidemiological studies, including analyses of outbreaks and hospital-to-hospital strain transmission events. Well-known advantages of typing methods based on PCR (low cost, short time, and easiness of performance) make MLVA a method that may be useful in a variety of laboratories, including those performing routine microbiological analyses within medical centers.

Countrywide Spread of CTX-M-3 Extended-Spectrum β-Lactamase-Producing Microorganisms of the Family Enterobacteriaceae in Poland

ABSTRACT Eighty-four clinical isolates of the family Enterobacteriaceae, recovered from 1998 to 2000 in 15 hospitals in 10 Polish cities, were analyzed. All the isolates produced β-lactamases with pIs of 8.4 and 5.4, and the pI 8.4 enzymes were demonstrated to hydrolyze cefotaxime but not ceftazidime in the in vitro bioassay. PCR analysis and DNA sequencing have revealed that in all cases the pI 8.4 β-lactamase was probably the CTX-M-3 extended-spectrum β-lactamase (ESBL) variant, which was originally identified in 1996 in Praski Hospital in Warsaw. In the majority of isolates, blaCTX-M-3 genes resided within large conjugative plasmids with similar fingerprints, which, in the context of the high degree of diversity of the randomly amplified polymorphic DNA types of the isolates, suggested that horizontal transfer of plasmids was likely the main mechanism of CTX-M-3 spread. The dissemination of plasmids was probably preceded by the center-to-center transmission of several strains, as indicated by the identification by pulsed-field gel electrophoresis of closely related or possibly related Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii isolates in five different hospitals. CTX-M-3-producing organisms revealed a very high degree of diversity in β-lactam resistance levels and patterns. This was attributed to several factors, such as the production of other β-lactamases including additional ESBLs, possible quantitative variations in CTX-M-3 expression, segregation of AmpC derepressed mutants, and permeability alterations.

Complete Nucleotide Sequence of the pCTX-M3 Plasmid and Its Involvement in Spread of the Extended-Spectrum β-Lactamase Gene blaCTX-M-3

ABSTRACT Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum β-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides blaCTX-M-3, it also bears the blaTEM-1, aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of β-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only blaCTX-M-3, accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE): a prospective, multinational study

BACKGROUNDnGaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it difficult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the first structured survey on the occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in European hospitals.nnnMETHODSnNational expert laboratories recruited hospitals with diagnostic capacities, who collected the first ten carbapenem non-susceptible clinical isolates of K pneumoniae or E coli and ten susceptible same-species comparator isolates and pertinent patient and hospital information. Isolates and data were relayed back to national expert laboratories, which made laboratory-substantiated information available for central analysis.nnnFINDINGSnBetween Nov 1, 2013, and April 30, 2014, 455 sentinel hospitals in 36 countries submitted 2703 clinical isolates (2301 [85%] K pneumoniae and 402 (15%) E coli). 850 (37%) of 2301 K pneumoniae samples and 77 (19%) of 402 E coli samples were carbapenemase (KPC, NDM, OXA-48-like, or VIM) producers. The ratio of K pneumoniae to E coli was 11:1. 1·3 patients per 10u2008000 hospital admissions had positive clinical specimens. Prevalence differed greatly, with the highest rates in Mediterranean and Balkan countries. Carbapenemase-producing K pneumoniae isolates showed high resistance to last-line antibiotics.nnnINTERPRETATIONnThis initiative shows an encouraging commitment by all participants, and suggests that challenges in the establishment of a continent-wide enhanced sentinel surveillance for carbapenemase-producing Enterobacteriaeceae can be overcome. Strengthening infection control efforts in hospitals is crucial for controlling spread through local and national health care networks.nnnFUNDINGnEuropean Centre for Disease Prevention and Control.

Molecular Characteristics of KPC-Producing Enterobacteriaceae at the Early Stage of Their Dissemination in Poland, 2008–2009

ABSTRACT After the first report in May 2008, the National Reference Center for Susceptibility Testing confirmed 113 cases of infection or colonization by KPC-producing members of the family Enterobacteriaceae in Poland by the end of 2009. The vast majority of patients were found in 18 hospitals; three patients were diagnosed at outpatient clinics. Most of the institutions were in the Warsaw area, including three hospitals with the highest numbers of cases. When available, the data on previous hospitalizations often indicated that these hospitals were the probable acquisition sites; one patient arrived from New York. The group of 119 unique isolates consisted of Klebsiella pneumoniae (n = 114), followed by Klebsiella oxytoca (n = 3), and Escherichia coli (n = 2). The K. pneumoniae isolates were dominated by the clone sequence type 258 (ST258) (n = 111); others were ST11 and ST23. The ST258 group was heterogeneous, with 28 pulsed-field gel electrophoresis (PFGE) subtypes, ∼25 plasmid profiles, and nine β-lactamase patterns differing by KPC variants (KPC-2 mainly), and SHV-12, CTX-M-3, and TEM-1-like enzymes. Plasmids carrying blaKPC genes varied in size (∼48 to 250 kb), structure, and conjugation potential. Transferable IncFIIK plasmids of ∼110 to 160 kb, probably pKpQIL or its derivatives, were observed in all K. pneumoniae clones and in K. oxytoca. Also prevalent were nontypeable pETKp50-like plasmids of ∼50 kb, found in K. pneumoniae ST258 and E. coli isolates (ST93 and ST224). Two K. pneumoniae-E. coli pairs from single patients might represent the in vivo transfer of such plasmids. The striking diversity of KPC producers at the early stage of dissemination could result from several introductions of these bacteria into the country, their multidirectional evolution during clonal spread, and transfer of the plasmids.