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Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, Regional Reference Centre for Microbiological Emergencies, 9 via G. Massarenti, 40138 Bologna, Italy
The identification of patients colonized or infected with carbapenemase-producing Enterobacteriaceae (CPE), in order to control and prevent the global spread of multidrug-resistant (MDR) pathogens.
Methods
From June 1 to June 15, 2012, eight Citrobacter freundii strains with reduced susceptibility to carbapenems were isolated from rectal swabs of hospitalized patients during active screening following the detection of a Klebsiella pneumoniae carbapenemase (KPC) -positive patient on the ward. All isolates were analyzed phenotypically and molecularly by PCR and sequencing. Genotype clustering was performed by multilocus sequence typing (MLST) analysis.
Results
The isolates showed high rates of multidrug resistance profile. A phenotypic assay for carbapenemase production suggested the presence of metallo-β-lactamase (MBL). The blaVIM-1 gene was detected in all imipenem-resistant C. freundii isolates. MLST showed that the C. freundii isolates shared the same sequence type (ST). Phylogenetic analysis revealed a strict relationship with an ST5 C. freundii isolate from a diarrhea patient in China.
Conclusions
Our findings showed that the active surveillance program for CPE was useful, not only for the detection of KPC-producers, but also to identify and control the spread of other MDR pathogens that could expand the spectrum of circulating MDR pathogens.
In recent years, the emergence and the very rapid spread of Enterobacteriaceae carrying carbapenem-hydrolyzing lactamases (carbapenemases) have been observed throughout the world, representing a serious threat to public health due to the great limitations of antimicrobial therapy.
Recently, several outbreaks of plasmid-acquired carbapenemase-producing Enterobacteriaceae (CPE) have been reported in Europe, suggesting an intense circulation of these microorganisms in this area.
Among the different types of carbapenemases, class A klebsiella pneumoniae carbapenemase (KPC) and class B (Verona integron-encoded metallo-b-lactamase (VIM), Imipenemase (IMP), New Delhi metallo-β-lactamase (NDM)) carbapenemases have represented the most common findings, while more recently class D oxacillinase-48 (OXA-48) has emerged.
Epidemiology and molecular characterisation of metallo-β-lactamase-producing Enterobacteriaceae in a university hospital intensive care unit in Greece.
Carbapenemase-producers are mainly identified among Klebsiella pneumoniae and Escherichia coli, while for other Enterobacteriaceae this type of resistance is still relatively uncommon.
Epidemiology and molecular characterisation of metallo-β-lactamase-producing Enterobacteriaceae in a university hospital intensive care unit in Greece.
Detection of the Klebsiella pneumoniae carbapenemase type 2 carbapenem-hydrolyzing enzyme in clinical isolates of Citrobacter freundii and K. oxytoca carrying a common plasmid.
Detection of the Klebsiella pneumoniae carbapenemase type 2 carbapenem-hydrolyzing enzyme in clinical isolates of Citrobacter freundii and K. oxytoca carrying a common plasmid.
Here, we describe an outbreak of blaVIM-1-producing C. freundii isolated from rectal swabs of colonized patients hospitalized in an Italian hospital.
2. Methods
2.1 Antimicrobial susceptibility testing and molecular analysis of C. freundii isolates
From June 1 to June 15, 2012, eight isolates of C. freundii were isolated from rectal swabs of patients hospitalized on a medical ward of a hospital located in the Bologna metropolitan area as a result of active screening following the detection of a KPC-positive patient on the same ward. Each C. freundii strain was isolated using a chromogenic selective medium (Brilliance CRE, Oxoid, UK). Routine biochemical identification and antimicrobial susceptibility testing were carried out using the Vitek2 semi-automated system (bioMérieux, France). Minimum inhibitory concentration (MIC) results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.
Carbapenem MIC values were confirmed by E-test. C. freundii isolates were further tested by a disk-diffusion synergy test as confirmatory/discriminatory carbapenemase phenotypic assay.
Ambretti S, Gaibani P, Berlingeri A, Cordovana M, Tamburini MT, Bua G, et al. Evaluation of phenotypic and genotypic approaches for the detection of class A and class B carbapenemases in Enterobacteriaceae. Microb Drug Resist 2012; in press.
A PCR assay for bla genes, including VIM, IMP, GIM, SIM, and SPM was performed in order to identify the specific MBL gene, and the amplicons obtained were analyzed in order to identify the specific bla gene.
Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France.
Results were compared and aligned with reference sequences using the online BLAST database and CLUSTAL W software.
2.2 Multilocus sequence typing (MLST)
To determine the genetic relationships between the C. freundii isolates, MLST based on seven housekeeping genes (aspC, clpX, fadD, mdh, arcA, dnaG, lypS) was performed, as described previously.
In July 2011, the health agency of the Emilia-Romagna region issued a specific surveillance protocol in order to improve the prevention and control of CPE.
The implementation of the regional guidelines by local hospitals requires extended infection control measures, including: isolation or cohorting of colonized/infected patients; strict contact precautions and reinforced hand hygiene; active surveillance by rectal swab cultures; screening of contact patients (when a positive case is detected); and, for patients with higher clinical and/or epidemiological risks, culture screening upon admission to the hospital.
3. Results
From June 1 to 15, 2012, eight C. freundii isolates were recovered from specimens of colonized patients. The isolates were resistant to all β-lactams, β-lactam/β-lactamase inhibitor combinations, and imipenem, while still susceptible to ciprofloxacin, colistin, tigecycline, trimethoprim–sulfamethoxazole, meropenem, and ertapenem (Table 1). The synergy test gave positive results for the meropenem disk in association with dipicolinic acid for all eight strains, suggesting the production of MBL. PCR analysis and nucleotide sequencing revealed that all these isolates carried the blaVIM gene. Sequence analysis of the eight cloned amplicons performed with BLAST software showed 100% homology with the blaVIM-1 sequences available in GenBank.
Table 1Results of antimicrobial susceptibility testing of VIM-1-producing Citrobacter freundii strains: MIC values (mg/l) and EUCAST susceptibility interpretation
The MLST assay revealed that all the C. freundii strains had identical allele profiles. Comparison of the seven housekeeping genes obtained from a previously described human C. freundii isolate in China revealed several nucleotide substitutions among the seven housekeeping genes (data not shown). In particular, the highest variability was identified in the fadD gene (which is known to be the most variable among these seven genes). In order to evaluate the genetic relatedness of these C. freundii strains with previously reported isolates, we inferred an evolutionary relationship by constructing a neighbor-joining tree using a concatenated sequence of the seven housekeeping genes obtained from different human C. freundii isolates, as shown in Figure 1. The tree showed strict clustering of the Italian C. freundii isolates with the ST5 isolated in China from a patient suffering from acute diarrhea.
as indicated by multilocus sequence typing data. The phylogenetic tree was inferred using the neighbor-joining (NJ) method based on the aligned concatenated sequences of the seven housekeeping genes obtained from the different sequence types (ST) and Citrobacter rodentium (used as the outgroup).
On June 1, 2012, the local infection control committee of one of the hospitals located in the Bologna metropolitan area, received information about the isolation of a KPC-positive K. pneumoniae strain from a patient who was hospitalized on a medical ward. The committee correctly suggested that the affected ward apply active surveillance for all potential contact patients. As a consequence, 42 rectal swabs were taken within 1 day. This screening activity showed the detection of four patients colonized with a VIM- producing C. freundii, while no other KPC-K. pneumoniae were found. During the following 2 weeks, four other patients from the same ward had a positive result for C. freundii, showing an identical phenotype. No clinically relevant isolates of these microorganisms were found among the patients hospitalized on the affected ward. Even though a detailed epidemiological investigation was performed, the evaluation of collected data did not permit the identification of the potential index case.
The cluster of colonized patients was managed by applying the measures suggested by the regional protocol: infection control operators visited the ward to investigate any incorrect care behavior and meetings to refresh and improve staff training were organized; all the colonized patients were subjected to cohorting to ensure the strict application of contact precautions; microbiological screening was performed to monitor the transmission of CPE to other patients. This early and intensive action allowed containment of the cluster and no more cases were detected starting from June 16, 2012. The screening activity was continued for an additional 4 weeks, and no more C. freundii bearing blaVIM-1 were identified.
4. Discussion
The global spread of Enterobacteriaceae carrying plasmid-mediated carbapenem resistance has been observed worldwide, but since 2010 the increase in these MDR organisms has been very rapid, especially in Europe.
Based on previous reports, the epidemiology of Enterobacteriaceae harboring different carbapenemase types, such as NDM and KPC, suggests a condition of endemicity in the Italian area.
Among the different carbapenemase-producing Enterobacteriaceae, the most common species is K. pneumoniae, followed by E. coli, while various others species have been isolated sporadically both from infected and colonized patients.
Epidemiology and molecular characterisation of metallo-β-lactamase-producing Enterobacteriaceae in a university hospital intensive care unit in Greece.
This study reports an outbreak of VIM-producing C. freundii in an Italian hospital. MLST analysis grouped all these eight C. freundii isolates in a close relationship with the ST5 strain recently reported from a diarrhea patient in China.
The rapid identification of patients colonized and infected with CPE is recognized as a basic tool to control and prevent the global spread of these MDR pathogens.
In our study, we showed that active surveillance performed by rectal swab culture screening of all the potential contacts is useful, not only to control inter-patient transmission of a previously known MDR strain, but also to detect additional colonization with other types of CPE with the potential to spread among hospitalized patients. This approach thus represents an efficient measure to minimize the risk of nosocomial outbreaks caused by carbapenemase producers. The accurate and rapid identification of VIM-producing C. freundii strains allowed the infection control team to rapidly detect a cluster of patients colonized with this unusual type of CPE. This quick identification allowed the medical ward to contain the dissemination of these MDR bacteria among the hospitalized patients. It is noteworthy that despite the unfavorable clinical features of most patients (mean age >84 years, many comorbidities), colonization had not given rise to any clinically significant infection. This favorable clinical outcome could be due to several factors, possibly including low bacterial loads and mild pathogenic capacities of the colonizing germs. In addition, it is not yet clear if certain types of CPE and/or certain types of carbapenemase could have a higher pathogenicity than others. Nevertheless the observation that none of the eight patients colonized by VIM-producing C. freundii had signs of clinical infection related to this MDR organism should not lead to the underestimation of the value of active surveillance measures, because any further spread to other patients might have resulted in more serious clinical effects.
In conclusion, our data confirm that in order to contain and control the spread of carbapenem-resistant Enterobacteriaceae, it is necessary to maintain and enforce all the infection control measures, among which a pivotal role is played by microbiological screening of potentially colonized patients.
Acknowledgements
We thank Dr Li Bai for providing us with the C. freundii and C. rodentium sequences from human clinical isolates from China.
Funding: The main funding for this work was obtained from Regione Emilia Romagna (fund ‘Lab P3’ to VS).
Conflict of interest: None declared.
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Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe.
Epidemiology and molecular characterisation of metallo-β-lactamase-producing Enterobacteriaceae in a university hospital intensive care unit in Greece.
Detection of the Klebsiella pneumoniae carbapenemase type 2 carbapenem-hydrolyzing enzyme in clinical isolates of Citrobacter freundii and K. oxytoca carrying a common plasmid.
Ambretti S, Gaibani P, Berlingeri A, Cordovana M, Tamburini MT, Bua G, et al. Evaluation of phenotypic and genotypic approaches for the detection of class A and class B carbapenemases in Enterobacteriaceae. Microb Drug Resist 2012; in press.
Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France.
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