Background
The emergence of plasmid-mediated carbapenem hydrolyzing β-lactamases and their spread amongst Gram-negative bacteria, especially
Klebsiella pneumoniae, has become a serious threat for hospitalized patients worldwide. The two principal types of acquired carbapenemases are the molecular class B metallo-β-lactamases (MBLs) and the molecular class A
K. pneumoniae carbapenemases (KPCs), both of which have been detected in many countries [
1‐
4]. The
bla
VIM gene encodes a transferable carbapenemase and is generally found within class 1 integrons. Although originally identified in
Pseudomonas aeruginosa, VIM enzymes are now endemic within
Enterobacteriaceae [
5,
6]. The
bla
KPC genes are usually located within
Tn4401 [
7], a
Tn3-based transposon harbored on plasmids or in the chromosome. In recent years carbapenem-resistant
K. pneumoniae has become a frequent cause of nosocomial infections in several hospitals in different Venezuelan cities [
8], but the epidemiology, molecular epidemiology, clinical impact and carbapenemase genes of Venezuelan carbapenemase-producing
K. pneumoniae have not been described. In this study we characterize 19 carbapenemase-producing
K. pneumoniae isolates associated with nosocomial infections centered in the pediatric service of a large Venezuelan tertiary-care public hospital. All 19 isolates contained both
bla
VIM-2 and
bla
KPC-2 genes, were phenotypically sensitive to QACs and contained
qacΔE and
addA2 genes typical of class 1 integrons. Analysis by REP-PCR and MLST showed that all isolates had identical profiles characteristic of sequence type ST833.
Discussion
We report 19 carbapenem reistant K. pneumoniae strains isolated from the pediatrics service of a hospital in Venezuela that co-harbor both bla
KPC and bla
VIM genes. All 19 strains appear to have identical Rep-PCR profiles, and by MLST all appear to belong to ST833, suggesting that this resistant strain has become endemic in the pediatric service, especially in the neonatal units of this hospital, where 12 of the strains were isolated. The limited patient information available and irreversibly anonymous nature of the strains did not permit retrospective analysis of the patients to exclude the possibility that some represented colonization or transient carrier states with KPC-producing K. pneumoniae. This seems unlikely, as 15 of the 19 strains were isolated from blood cultures, suggesting they were associated with severe infections. Cross contamination also seems unlikely as the isolates showed seven different profiles of resistance to other antibiotics.
There are no official data available about epidemiological monitoring of bacteria causing nosocomial infections in Venezuelan hospitals. The only surveillance program that provides data about antimicrobial resistance in Venezuela is PROVENRA (
http://provenra.cloudapp.net/), a private initiative with information on antibiotic resistance from 1998 to 2012. The information is collected from microbiology laboratories in 46 hospitals nationwide, but does not include the “Dr. Luis Razetti” hospital studied in the present report. According to data reported for 2012 on the PROVENRA webpage, 72 % of
K. pneumoniae isolated in neonatology services were resistant to ertapenem and 65 % were resistant to meropenem and imipenem (
http://provenra.cloudapp.net/). This high prevalence of resistance was confirmed in our study.
Other studies have described carbapenem resistance in Venezuela. Fernández-Canigia and Dowziki in 2012 [
31] presented data from the Latin American region on Gram-negative isolates used in the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.). They describe decreasing susceptibility to carbapenems among ESBL producing
K. pneumoniae in the Latin America countries studied, but found that 90.3 % of Venezuelan strains were sensitive to meropenem. Jones et al. [
32] reported the results of a resistance surveillance program monitoring antimicrobial susceptibility patterns in Latin America in which only 15 % of
Klebsiella isolates were carbapenem-resistant. Finally, Kazmierczak et al. [
33] analyzed Gram-negative pathogens collected from 40 countries, including Venezuela, as part of a global surveillance study in 2012–2014. Carbapenem non-susceptible
Enterobacteriaceae were characterized for
bla genes encoding MBLs and serine β-lactamases variants with PCR and sequencing. In the strains from Venezuela there was one isolate of
K. pneumoniae containing NDM-1 and one isolate of
P. aeruginosa carrying VIM-2 [
33].
The KPC-2 allele, one of 21 variants of the
bla
KPC gene, was found in all our 19
K. pneumoniae isolates and is one of the most extensively distributed worldwide [
34], including in the South American countries of Colombia [
35,
36] Brazil [
37‐
40] Argentina [
41,
42]. In Venezuela [
43,
44], a KPC-2-producing
K. oxytoca was isolated from a pediatric patient in the state of Mérida [
44], but to our knowledge, KPC genes have not been previously reported in Venezuelan
K. pneumoniae isolates.
In addition to the KPC-2 gene, all of our 19 isolates also carried the VIM-2-carbapenemase. The
bla
VIM gene is extensively distributed worldwide, with VIM-2 the most widespread variant [
45]. Endemicity of VIM enzymes has been reported in Greece, Taiwan, and Japan [
46,
47], although outbreaks and single strains of VIM producers have been reported in many other countries including the Latin American countries of México, Argentina, Colombia and Venezuela [
45]. In Venezuela the
bla
VIM gene was previously found in clinical isolates of
Pseudomonas aeruginosa [
48,
49], and Marcano et al. [
50] reported VIM-producing carbapenem-resistant
K. pneumoniae isolated from the urine of a 7-year-old girl hospitalized at the “Hospital de Niños J. M. de los Ríos” in Caracas, Venezuela.
Strains carrying both
bla
KPC and
bla
VIM carbapenemases have been previously reported in Greece [
1,
51‐
54] Colombia [
55], Germany [
56], Italy [
6], and Spain [
57]. In Venezuela the two carbapenemases have only been reported together in a multiply resistant strain of
Enterobacter cloacae [
58] isolated from the urine of a 83-year-old patient in a hospital in the city of Cumaná, which is only about 90 miles from hospital we studied. It might be interesting to know whether both the
E. cloacae and
K. pneumoniae isolates could be carrying these carbapenemase genes within the same transferable genetic context.
Class 1 integrons are the most common integron type present in clinical isolates of the
Enterobacteriaceae, and are increasingly detected in isolates of
K. pneumoniae. The gene cassettes most frequently identified within class 1 integrons in
Enterobacteriaceae are those encoding resistance to streptomycin (
aadA) and trimethoprim (
dfrA) [
59,
60]. By PCR we found that 19
K. pneumoniae strains contained the
addA2 gene within the 5′CS-3′CS region of the integron. We did not look for the presence of the
dfrA gene, but all 19 strains were resistant to trimethoprim-sulfamethoxazole. The
qacΔE gene, which has also been associated with class 1 integrons [
61] was amplified from all 19 isolates.
KPC genes are often found within a transposon-associated element,
Tn4401 [
7].
Tn4401 possesses genes encoding a transposase (
tnpA) and a resolvase (
tnpR), and has been characterized as an active transposon that is able to mobilize the
bla
KPC genes at high frequency without target specificity [
7,
25]. In all 19 strains in this study the
bla
KPC-2 genes appeared to be within a
Tn3-based structure consistent with the
Tn4401 isoform ‘b’ [
62], which has been observed in Greece [
63], Colombia [
14], Brazil [
64] and the USA. Similar to the report by Pereira et al. [
64] the inverted repeat sequences of the flanking regions were not amplified in our isolates, suggesting that their insertion sites may be different from those of
K. pneumoniae YC described by Naas et al. [
7].
The KPC and VIM genes are generally found on plasmids [
65], and the plasmids profiles from all 19 strains contained similar bands, suggesting that the integron containing the carbapenemase genes could be present within a transposable element on a common plasmid, but further studies are needed to confirm this possibility.
All 19 isolates evaluated in this study demonstrated similar patterns with REP-PCR analysis, and by MLST all belonged to ST833, a genotype that has only been reported in Israel [
28] and Trieste, Italy [
29]. Interestingly, the ST833
K. pneumoniae strain isolated in the Trieste Pediatric Hospital was from a blood culture of a 3-year-old patient transferred from a Venezuelan hospital to undergo marrow transplantation [
29].
We have not found any previous studies describing MLST characterization of K. pneumoniae strains isolated in Venezuela, but we have used MLST to analyze the strains of KPC-producing K. pneumoniae in a few other hospitals in the country (manuscripts submitted). Although the other hospitals always contained a variety of sequence types, we found isolates belonging to ST833 in hospitals in two other Venezuelan states--Zulia and the Capital District. These are in the west and center of the country, respectively, and distant from hospital studied in the current report, which is located in the eastern state of Anzoátegui. From our albeit limited sampling, it appears that the carbapenem resistant K. pneumoniae ST833 strain could be extensively distributed throughout the country. Perhaps a Venezuelan patient acquired the ST833 strain as an infection or colonization while hospitalized in Israel and then returned to Venezuela, where it subsequently disseminated throughout the country with the movements of patients and health care staff.
ST833 is part of the 258 clonal complex, and the ST833 allelic profile (3-3-1-1-1-1-12) differs from ST258 only in the
tonB allele. The clonal complex CC258 is the dominant and most successful KPC producing strains as has spread widely and rapidly across the world [
30,
66], although the reasons for its apparent advantage have yet to be completely explained facilitated by its production of proteins involved in cell motility, secretion and DNA repair and modification [
67‐
69].
Disinfectants containing QACs are commonly used in Venezuelan hospitals, and therefore the presence of QAC resistance would complicate efforts to reduce the prevalence of this ST833 strain. Fortunately, all strains were phenotypically sensitive to the QAC disinfectant and we could not amplify the
qacA,
qacB [
15] or
qacC genes [
16,
17] which have all been associated with QAC resistance. Nevertheless, the use of QAC disinfectants is clearly only a minor part of the measures that are required to control nosocomial infections.
Venezuelan medical personnel are keenly aware that carbapenem-resistant
K. pneumoniae have become a frequent cause of nosocomial infections in several hospitals in different Venezuelan cities [
8]. We hope that the molecular epidemiology provided by this study can aid in tracking the presence and dissemination of the strains involved, and thereby contribute to the vigilance and surveillance required to effectively treat them and to reduce their presence.