Patients and infections.
During 2009, CNSKP isolates were obtained from clinical samples from 55 patients admitted to either the SICU (n = 36, 65.5%) or the MICU (n = 19, 34.5%). Of the 55 patients, 28 (50.9%) were infected by CNSKP, while 27 (49.1%) had no clinical evidence of infection and were considered colonized. The incidence of new cases in the SICU was significantly higher than that in the MICU (16.7 versus 6.1 cases/1,000 patient-days) (relative risk, 2.7; 95% CI, 1.6 to 4.7; P < 0.001). Thirty-five (63.6%) patients were male. Twenty-one (38.2%) were >65 years old, and 34 (61.8%) were >18 and ≤65 years old. The median MICU/SICU stay was 36 days (range, 3 to 101 days).
The clinical characteristics, treatments, and outcomes of the patients infected by CNSKP are detailed in . The most prevalent infections diagnosed were pneumonia and catheter-associated bacteremia, with seven cases (25%) each. Of the 27 patients colonized, CNSKP was recovered from exudates of the rectum (22 cases; 78.6%), trachea (3 cases; 11.1%), urethra (1 case; 3.7%), and bladder catheter (1 case; 3.7%).
Clinical characteristics of patients infected with VIM-1-producing Klebsiella pneumoniaea
Of the 55 patients studied, 45 (81.8%) had previous underlying conditions, and 25 of them (55.6%) had more than one. The most prevalent underlying diseases were neoplasia (13 cases; 25.5%), immunosuppression due to solid organ transplantation (12 cases; 21.8%), and heart diseases (21 cases; 38.2%).
Thirteen (46.4%) infected patients and 11 (40.7%) colonized patients, all of them with severe underlying diseases, died.
Twenty-two of the 28 (78.6%) infected patients were treated with antibiotics active against CNSKP, mainly tigecycline in monotherapy (9 cases; 32.1%) or in combination with colistin (11 cases; 39.3%) (). Of the remaining 6 infected patients, 2 were treated with meropenem or ertapenem because their isolates' MICs for both antibiotics were 1 μg/ml and interpreted to be susceptible according to previous CLSI criteria (ertapenem MIC ≤ 2 μg/ml and meropenem MIC ≤ 4 μg/ml; document M100-S20 [7a
]), 2 did not receive treatment due to early death, and the clinical condition of 2 patients improved after central venous catheter or bladder catheter removal ().
Nineteen of the 28 patients infected (67.9%) received adjuvant treatment, mainly change or removal of the central venous catheter (14 patients) ().
Susceptibility testing of CNSKP isolates.
During the study period, 335 CNSKP isolates were recovered from different samples from the 55 patients studied. All of them were resistant to ampicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, piperacillin-tazobactam, gentamicin, tobramycin, ciprofloxacin, and co-trimoxazole; 36% were susceptible to amikacin. The MICs for tigecycline ranged from 0.25 μg/ml to 4 μg/ml (MIC50 = 0.5 μg/ml, MIC90 = 1 μg/ml). All isolates were susceptible to colistin (MICs < 2 μg/ml).
Imipenem, meropenem, and ertapenem MICs ranged from 1 to >8 μg/ml (MIC50 and MIC90, >8 μg/ml in all cases). The modified Hodge test with imipenem or imipenem-EDTA Etest strips was positive for all CNSKP isolates.
In total, 99 representative CNSKP isolates were subjected to further molecular epidemiology studies. PFGE results revealed two well-defined clusters. Cluster 1 (C1) was predominant in both infected (n = 27) and colonized (n = 27) patients, while isolates of cluster 2 (C2) were isolated from only four patients, although only one was infected; the three patients colonized by isolates of C2 were also infected (n = 1) or colonized (n = 2) by isolates of C1.
According to the minor banding pattern differences found, C1 could be subdivided into four subtypes, C1A to C1D; two subtypes were also identified in C2, C2A and C2B (). By MLST analysis, C1 and C2 were identified to be sequence type 15 (ST15) (six isolates tested, three of subtype C1A and one each of C1B, C1C, and C1D) and ST340 (three isolates tested, two of subtype C2A and one of C2B), respectively (). This is the first description of VIM-1-producing K. pneumoniae
ST15, an MLST type previously associated with CTX-M-15-producing epidemic clones in Hungary and Denmark (11
). ST340 is a single-locus variant of the widely disseminated KPC-producing ST258 strain (18
Fig 1 Dendrogram illustrating the PFGE profiles and MLST types of the two different clones of VIM-1-producing Klebsiella pneumoniae isolates and the number of patients infected and/or colonized by each. One patient was infected by clone C1/ST15 and colonized (more ...) Antibiotic resistance genes and class 1 integron characterization.
blaVIM-1 was identified in both the C1/ST15 and C2/ST340 clones. blaVIM-1 was carried in a class 1 integron in the following cassette combination: intI1 (integrase gene)–blaVIM-1–aac(6′)-Ib (tobramycin resistance gene, also called aacA4)–dhfrII (trimethoprim resistance gene)–aadA1 (streptomycin resistance gene)–catB2 (chloramphenicol resistance gene)–qacEδ1/sul-1 (quaternary ammonium compound resistance gene/sulfonamide resistance gene).
C1/ST15 isolates also had the blaSHV-134
gene encoding the new ESBL SHV-134 (http://www.lahey.org/studies/webt.asp
has a single nucleotide change at position 448 (C → G), in comparison with the sequence of blaSHV-12
, which codes for an amino acid substitution at position 154 (Q → E) (GenBank accession number HM559945
). The insertion sequence IS26
was detected linked 73 bp upstream of blaSHV-134
, as previously described in the blaSHV-12
). This fact supports the idea of genetic evolution of blaSHV-134
from the blaSHV-12
The blaTEM-1 gene and the aminoglycoside resistance gene aac(3′)-IIa were also identified in K. pneumoniae isolates representative of both clones.
A selected sample of four K. pneumoniae isolates representing the different clones (two C1/ST15 isolates and two C2/ST340 isolates) with MICs of >8 μg/ml to imipenem, meropenem, and ertapenem was studied further to determine ompK35 and ompK36 sequences. In the two C1/ST15 isolates, the DNA sequences of ompK35 and ompK36 showed point mutations at positions 690 (G → A) and 360 (C → A), generating TGA and TAA premature stop codons, respectively. In the two C2/ST340 isolates, the sequence of ompK35 had a point mutation at position 520 (C → T), generating a TAG premature stop codon, whereas no changes were detected in the ompK36 sequence.
Conjugation assay and plasmid characterization.
Carbapenem-nonsusceptible E. coli
transconjugants were obtained from isolates of the C1/ST15 and C2/340 clones (). All transconjugants carried a plasmid of ~50 kb that was untypeable by PCR and from which positive identification of the blaVIM-1
gene and a class 1 integron was obtained (). Untypeable plasmids of a similar size were found in a recent study by Miró et al. (25
). Previous European studies had detected blaVIM-1
in association with plasmids of incompatibility groups N (32
), I (35
), and HI2 (25
Antibiotic susceptibilities of VIM-1-producing Klebsiella pneumoniae isolates K534 of clone C1/ST15 and K535 of clone C2/ST340 and their transconjugants, TC1K534, TC2K534 and TC1K535
Molecular characteristics of VIM-1-producing Klebsiella pneumoniae isolates K534 of clone C1/ST15 and K535 of clone C2/ST340 and their transconjugants, TC1K534, TC2K534 and TC1K535
In addition, cefotaxime-resistant but carbapenem-susceptible transconjugants were also obtained from isolates of the C1/ST15 clone that were TEM-1 and SHV-134 producers and carried a single IncFIIA plasmid of ~75 kb ( and ). Simultaneous production of VIM-1 and ESBLs of the SHV family has previously been described in K. pneumoniae
), but this is the first report of the novel ESBL SHV-134 and VIM-1. blaSHV-134
is located in a conjugative IncFII plasmid other than the plasmid harboring the blaVIM-1
gene. Previous reports described the association of blaSHV-12
with IncFII plasmids (12
Case-control clinical study.
All the 55 patients infected and/or colonized by VIM-1-producing K. pneumoniae isolates and 55 control patients were included in the study. No differences between case and control patients regarding age, gender, and frequency of surgery could be found ().
Univariate analysis of risk factors linked to infection or colonization by VIM-1-producing Klebsiella pneumoniae
The total hospital stay was longer for cases than for controls (median stay, 36 versus 7 days; P < 0.001), as was the Apache II index (21.4 versus 18.8; P = 0.08). No significant differences in frequency of underlying diseases were observed between the two groups; however, the case-fatality ratio was significantly higher for case patients than for control patients (45.5% and 30.9%, respectively; P = 0.003).
In general, infection or colonization by VIM-1-producing K. pneumoniae was significantly associated with total previous exposure to invasive devices and a lengthened stay in the ICU (P < 0.001). Previous exposures included, in particular, central and peripheral venous catheters, mechanical ventilation, a nasogastric tube, parenteral nutrition, and a tracheotomy. Cases also received more antibiotics and received antibiotics for longer periods of time than their control counterparts (). Furthermore, use of quinolones, carbapenems, piperacillin-tazobactam, linezolid, and extended-spectrum cephalosporins for more than 7 days was associated with a greater risk of acquiring CNCKP (). The main independent risk factors identified by the general multivariate model were the number of days that the patient received mechanical ventilation and the use of parenteral nutrition; previous treatment with linezolid and extended-spectrum cephalosporins for more than 7 days was identified as a risk factor by the antimicrobial use model ().
Potential risk factors associated with VIM-1-producing Klebsiella pneumoniae colonization or infection as determined by multivariate analysisa
Taking into account the fact that the size of this observational study precludes the identification of several independent factors, which would otherwise be interrelated, the factors that were identified indicated that very sick patients who required intensive care for long periods of time was at greater risk for infection or colonization by VIM-1-producing K. pneumoniae. These factors give greater opportunities to acquire bacteria, especially in an environment where intensive antibiotic treatments select those species resistant to many antibiotics. One of the interesting findings in this observational study is the independent association of a previous exposure of cases to linezolid. On the one hand, it could be signaling those patients who developed more severe infections and were empirically treated for infections with Gram-positive microorganisms, but, on the other hand, linezolid could be helping to eradicate patients' Gram-positive bacterial flora, thereby allowing colonization by resistant Gram-negative bacteria.
Epidemic curve and infection control measures.
The epidemic curve of patients infected by CNSKP in the two ICU units is displayed in . Almost all the MICU cases appeared in the second half of the year, from week 27 to year end. The dissemination of CNSKP from the SICU to the MICU during the summer period was linked to the fact that the MICU was closed; therefore, MICU patients were allocated to one of the SICU modules and nurses, registered nurses, warden, and cleaning personnel were shared by MICU and SICU patients. This circumstance allowed dissemination of the bacteria between the two populations, indicating that contact transmission is a key factor for the spread of the epidemic clone.
Epidemic curve of cases of infection by VIM-producing Klebsiella pneumoniae in two intensive care units. SICU-I, infected patients in surgical intensive care unit; MICU-I, infected patients in medical intensive care unit.
Infection control personnel identified all patients with VIM-1-producing K. pneumoniae isolates recovered from any clinical specimen according to the microbiology laboratory reports. In addition, active surveillance of all patients admitted to the ICUs was performed once a week. Patients identified to be harboring VIM-1-producing K. pneumoniae were assigned to contact precautions, including a single room, when available, or patient cohorting and the use of gowns and gloves that were discarded after caring for a patient. In addition, standard precautions were reinforced for all patients admitted to the ICUs, including improvement of hand hygiene compliance through the use of alcohol rubs before and after caring for patients. The outbreak described here did not stop during 2010 (data not shown) but remained at a lower incidence toward endemicity (incidence rate, 4.4 cases/1,000 patient-days in 2010). Eradication of these bacteria in this vulnerable population can be very difficult to achieve, in spite of drastic measures, such as an aggressive infection control strategy.
In this outbreak, we observed three periods without cases of infection: between weeks 10 and 16, 31 and 33, and 36 and 39 (). These periods followed both the educational series in the SICU on week 9 and the direct observation of the personnel and the requirement to comply with basic hand hygiene and contact precaution measures on weeks 28 to 29 and 36 to 37 for the SICU and MICU, respectively (). The latter intervention is costly and could not be continued due to economic constraints. Other potentially effective but costly interventions, which include the cohorting of carriers and staff or even closing of the units and the total removal of all environmental as well as patient reservoirs, were not undertaken (23