Abstract
Background
Klebsiella infections are reported from neonatal intensive care units (NICUs) worldwide, but data on their incidence and genetic diversity remain scarce.
Objective
We determined the incidence and genetic diversity of Klebsiella infections in NICU patients in Rio de Janeiro.
Methods
This was a prospective study including newborns admitted to NICU in three hospitals during April 2005–November 2006 and March 2008–February 2009. Klebsiella pneumoniae isolates were genotyped by multilocus sequence typing (MLST) and extended spectrum β-lactamases (ESBL) were characterized.
Results
Klebsiella infections occurred in 38 of 3984 patients (incidence rate, 9.5/1000 admissions); 14 (37%) of these 38 newborns died. Two clonal groups, CC45 and CC1041, caused 11 cases (42% of K. pneumoniae infection). Ten (32%) of the isolates causing infection produced ESBL, 9 of which (83%) carried blaCTX-M-15, all belonging to clonal complex (CC) 45 and CC1041. Nine of these ESBL-producing isolates were confined to only one of the NICUs.
Major conclusions
The high incidence of Klebsiella infections in NICU in Rio de Janeiro appeared to be due to a combination of frequent sporadic infections caused by multiple K. pneumoniae genotypes and small outbreaks caused by dominant multidrug-resistant clones.
Keywords: Klebsiella, Bloodstream infection, Multilocus sequence typing, Newborn
Introduction
Klebsiella infections affect mainly immunocompromised patients and those requiring invasive medical procedures such as mechanical ventilation [1]. Newborns who require admission to neonatal intensive care units make up a population particularly vulnerable to these infections [2–4], owing to the combination of the reduced functional capacity of the immature neonate immune system and the frequent exposure to invasive procedures. Relative frequency of Klebsiella sp. infections ranges from 21 to 52% of all infections in newborns admitted to neonatal intensive care units (NICUs) due to multidrug-resistant (MDR) Gram-negative bacteria in Latin America [5]. A major current concern is the dissemination of strains expressing extended spectrum β-lactamases (ESBL), Klebsiella pneumoniae carbapenemase (KPC), and New Delhi metallo-beta-lactamase (NDM). ESBL- and KPC-producing K. pneumoniae became endemic, while NDM is emerging in many places in Latin America [6, 7]. In 2014, 19–87% of K. pneumoniae isolates were reported to be ESBL-producing strains in this region [8]. The blaKPC gene was first reported in Brazil in 2005 and described throughout the country afterwards [7]. Strains expressing CTX-M-type ESBL have disseminated worldwide, and CTX-M-15 became one of the most common ESBL variants in Klebsiella [9–11]. This ESBL sub-type has been described in Klebsiella isolates from newborns, causing infection or just colonization [12], but is not limited to hospitals [13]. In recent years, the isolation of CTX-M-15-producing isolates has been increasingly reported in community settings as asymptomatic colonization of humans or in the environment [13]. In Brazil, CTX-M-15-producing K. pneumoniae [14] belonging to multilocus sequence type (MLST) 1, 11, 14, 17, 20, 35, 36, 147, and 490 have been reported to cause bloodstream infections (BSI) in adults. These strains are frequently recovered globally from newborns and adults, causing either infection or colonization [12, 15, 16]. Nevertheless, there is paucity of information about the population structure of isolates from newborns, as most published reports typically describe hospital infections and outbreaks caused by multidrug-resistant K. pneumoniae [2–4]. The increasing incidence of these infections, especially in hospitals in large urban settings, suggests that Klebsiella organisms have become endemic in healthcare environment. The aim of the present study was to determine the incidence of Klebsiella infections among newborns admitted to three intensive care units (NICUs) of the Municipal Health System of Rio de Janeiro and to investigate whether these infections were polyclonal and represented epidemiologically unrelated infections or outbreaks.
Material and methods
Study design
Between April 2005 and November 2006, and between March 2008 and February 2009, we prospectively identified Klebsiella infections and newborns colonized in the NICU of three public hospitals (A, B, and C) of the municipal health system of Rio de Janeiro. We included newborns hospitalized in the NICU for > 24 hours (h), followed from admission to discharge, death or transfer to another unit. Only the first admission was considered. Admission of patients to NICU occurred on the date of birth. Patient data were collected prospectively by infectious disease attending physicians, and included demographic data, use of invasive devices, and presence of clinical infections diagnosed as proposed by the National Healthcare Safety Network (NNIS), CDC. We considered antimicrobial therapy appropriate when a drug to which the organism was susceptible was started within 48 h of diagnosis of infection. We classified death as related to infection according to the judgment of a neonatologist member of the study team, who prospectively evaluated all cases. Infections were considered related to death when uncontrolled until the outcome, or, if controlled, the consequences were enough to cause death; infections were considered unrelated to death when the underlying disease morbidity was significantly greater in severity than the infectious process and could directly cause a lethal outcome.
Bacterial isolates
Bacterial isolates consisted of those obtained consecutively during investigation of infectious episodes diagnosed at NICUs. Infection isolates were recovered from blood (26; 79%), urine (5; 15%), catheter (1; 3%), and cerebrospinal fluid (1; 3%). Additionally, for comparison purposes, we included isolates obtained from specimens collected for surveillance of colonization or discarded as infection causes, by ESBL-producing organisms. Most of those isolates were recovered from rectal swabs (40; 88%); others were from tracheal aspirate (1; 2%), blood (1; 2%) (infection discarded by attending physician), pyloric secretion (1; 2%), urethral meatus (1; 2%), umbilical secretion (1; 2%), and ocular secretion (1; 2%). Such surveillance practice had no uniform policy among hospitals. We studied a convenience sample of these colonization isolates to compare the genotypes of K. pneumoniae clonal types isolated from infection cases. During the study, 79 isolates from colonization and infection recovered from 66 newborns were identified by the analytical profile index (API) (API20E (Enterobacteriaceae)) system in the original laboratories of each hospital as Klebsiella spp. These were the isolates included in this study.
To confirm identification of isolates, we performed a panel of classical phenotypic tests for Enterobacteriaceae [17], in addition to growth at 10 °C, histamine and D-melezitose assimilation. Species ID was confirmed by rpoB gene sequence analysis [18]. For isolates identified as Raoultella, we sequenced both DNA strands of blaORN and rrs (which encodes16S rDNA) [19, 20].
Susceptibility was determined by disk diffusion test [21] to the following antimicrobial agents: amikacin, amoxicillin/clavulanic acid, ampicillin, aztreonam, cephalothin, cefepime, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, meropenem, and trimethoprim/sulfamethoxazole. ESBL production was detected by double diffusion test with ceftazidime, cefotaxime, cefepime, and aztreonam disks applied at 2.5 cm from amoxicillin/clavulanic acid [22].
The presence of blaCTX-M, blaKPC, and blaNDM genes was tested by PCR in ESBL-producing isolates in a volume of 25 μl containing 2.5 μl of 10x Taq DNA polymerase buffer, 0.2 mM of each dNTP, 2 mM MgCl2, primers 10 pmol/μl, 0.65 U of Taq DNA polymerase, and 1 μl of the DNA template. Amplification conditions were initial denaturation at 95 °C for 1 min, annealing at 57 °C for 1 min, extension at 72 °C for1 min, and final extension at 72 °C for 7 min.
To locate ESBL genes on plasmids, we performed conjugation experiments to transfer blaCTX-M gene by plasmid transfer mating-out assays using azide-resistant Escherichia coli J53 as recipient strain. We selected transconjugants on 150 μg/ml sodium azide and 2 μg/ml ceftriaxone final concentration. Analysis of transconjugants included PCR for blaCTX-M gene and disk-diffusion susceptibility tests for β-lactam and non-β-lactam agents.
Strains were initially subtyped by ERIC-PCR as previously described [23]. Amplification conditions were as follows: initial denaturation at 94 °C for 30 s, annealing at 54 °C for 1 min, extension at 72 °C for 4 min, and final extension at 72 °C for 1 min. We analyzed electrophoretic banding profiles with Bionumerics, version 7.1 (Applied Maths, Kotrijk, Belgium). We constructed dendrograms based on Dice index and unweighted pair group method with arithmetic average (UPGMA).
Multilocus sequence typing (MLST) was performed as previously described [24]. PCR used a total volume of 25 μl containing 2.5 μl of 10x Taq DNA polymerase buffer, 100 μM of each dNTP, 3 mM MgCl2, 0.3 μM of primer, and 1.5 U of Taq DNA.
All data were entered into a database built in Access (Microsoft Corp, Washington, EUA); differences between proportions were compared by the chi-square or Fisher’s exact test with OpenEpi platform [25].
Ethics
The study was approved by the ethics committees of the Brazilian institutions Hospital Universitário Clementino Fraga Filho (Protocol No. 235/04) and of Secretaria Municipal de Saúde of Rio de Janeiro (Protocol No. 75/08).
Results
After exclusion of 29 patients admitted > 24 h after birth, the final patient population consisted of 2245 newborns in the first and 1739 in the second study period, as detailed in Fig. 1. Among 3984 newborns, 333 (84/1000 admissions) had infections with microbiological confirmation during the study. Gram-positive cocci were isolated in 197 (59% of the infections, 49/1000 admissions), Gram-negative bacteria in 115 (35%, 29/1000 admissions), and fungi in 46 (14%, 12/1000 admissions). We observed 11 polymicrobial infections. Klebsiella caused 38 (11%; 9.5/1000 admissions) of these infections (Fig. 1), being the leading Gram-negative agent causing neonatal infection in this cohort.
Fig. 1.
Patient population and Klebsiella sp. isolates included in study
In total, 79 Klebsiella isolates, 33 (42%) from infection and 46 (58%) from colonization, were recovered from 66 newborns admitted to NICU, as described in Fig. 1. The 33 isolates from 32 infections were K. pneumoniae (82%), K. oxytoca (15%), and K. variicola (3%). The K. variicola isolate was obtained from a blood specimen containing also K. pneumoniae isolate. The 46 colonization isolates included K. pneumoniae (78%), K. oxytoca (14%), K. variicola (4%), and Raoultella ornithinolytica (4%). ESBL production was observed in 10 (30%) of infection and 33 (72%) of colonization isolates. Resistance to carbapenems was not detected.
Most infections occurred in male newborns (55%), with birth weight > 1500 g (60%), and born by vaginal delivery (53%), as shown in Table 1. Twenty (53%) of 38 patients did not receive any invasive device; use of devices was documented in more than half of newborns with infection by K. pneumoniae and absent in those with K. oxytoca infection (p = 0.06). Mechanical ventilation was performed on 12 (67%) of the 18 patients with infection by K. pneumoniae.
Table 1.
Analysis of newborns with Klebsiella infection: demographic and clinic characteristics of patients with infection by K. pneumoniae vs K. oxytoca
| Variable* |
Klebsiella pneumoniae (n = 33) |
Klebsiella oxytoca (n = 5) |
Total N = 38 |
|---|---|---|---|
| Male | 19 (58) | 2 (40) | 21 (55) |
| Weight ≤ 1.500 g | 13 (39) | 2 (40) | 15 (40) |
| Delivery | |||
| Vaginal | 17 (52) | 3 (60) | 20 (53) |
| Cesarean | 14 (42) | 2 (40) | 16 (42) |
| Unknown | 2 (6) | 0 | 2 (5) |
| APGAR: median (interquartile range) | 8 (7–9) | 8 (7–9) | 8 (7–9) |
| Gestational age: median (interquartile range) | 35 (30–38) | 34 (32–38) | NA |
| Diagnosis | |||
| BSI and meningitis | 27 (82) | 4 (80) | 31 (82) |
| Other** | 6 (18) | 1 (20) | 7 (18) |
| Presence of invasive device | 18 (55) | 0 | 18 (47) |
| Type of invasive device | |||
| CVC | 7 (26) | 0 | 7 (18) |
| PICC | 2 (7) | 0 | 2 (5) |
| Mechanical ventilation | 12 (44) | 0 | 12 (32) |
| Bladder catheter | 1 (4) | 0 | 1 (3) |
| TPN | 5 (18) | 0 | 5 (13) |
| Appropriate therapy | 12 (44)*** | 3 (60) | 15 (55) |
| ESBL producing | 9 (33)*** | 3 (60) | 12(44) |
| Death | 14 (42) | 0 | 14 (37) |
| Death related to infection | 7 (21) | 0 | 7 (18) |
Data are number of patients and (%) unless otherwise specified.**Including UTI, urinary tract infection [6], skin and soft tissue infection [10]. ***Results known for 27 of the 33 patients with isolate stored for further analysis. CVC, central venous catheter (umbilical catheter and venous dissection); PICC, peripheral insertion central catheter; TPN, total parenteral nutrition; ESBL, extended spectrum beta-lactamase. NA, not applicable. All comparisons are p > 0.05
ESBL production and appropriate therapy were determined for the 32 infections in patients whose isolates were stored for further analyses (27 of 32 K. pneumoniae and all 5 K. oxytoca). Treatment was appropriate for 15 patients: two (20%) of 10 infected with ESBL-producing strains and 13 (59%) of 22 infected with ESBL-non-producing strains (p = 0.09).
From the 38 patients infected by Klebsiella, 14 (37%) died; seven deaths were related to K. pneumoniae infection (21% of the patients with K. pneumoniae infection), while no newborns with K. oxytoca died (p = 0.16) (Table 1). The distribution of the characteristics of patients according to their outcome is shown in Table 2. Mechanical ventilation and total parenteral nutrition were administered to 71% and 43% of newborns who died, respectively.
Table 2.
Analysis of newborns with Klebsiella infection: demographic and clinic characteristics of patients with death related to infection vs discharge or death unrelated to infection
| Variable | Death related to Klebsiella infection (n = 7) | Discharge or death unrelated to Klebsiella infection (n = 31) |
|---|---|---|
| Male | 3 (43) | 18 (58) |
| Weight ≤ 1.500 g | 4 (57) | 11 (35) |
| Delivery | ||
| Vaginal | 5 (71) | 15 (48) |
| Cesarean | 2 (29) | 14 (45) |
| Unknown | 0 | 2 (6) |
| APGAR: median (interquartile range) | 7 (5–8) | 8 (7–9) |
| Gestational age: median (interquartile range) | 32 (30–36) | 35.4 (31–38) |
| Diagnosis | ||
| BSI and meningitis | 7 (100) | 25 (81) |
| Other* | 0 | 6 (19) |
| Invasive device | 6 (86) | 12 (39) |
| Type of invasive device | ||
| CVC | 2 (29) | 5 (15) |
| PICC | 1 (14) | 1 (3) |
| Mechanical ventilation** | 5 (71) | 7 (21) |
| Bladder catheter | 0 | 1 (3) |
| TPN*** | 3 (43) | 1 (3) |
| Appropriate therapy | 1 (14) | 15 (48) |
| ESBL-producing isolate | 2 (29) | 10 (32) |
| Age in days at diagnosis: median (interquartile range) | 19 (8–27) | 35 (8–56) |
CVC, central venous catheter (umbilical catheter and venous dissection); PICC, peripheral insertion central catheter; TPN, total parenteral nutrition; ESBL, extended spectrum beta-lactamase. *Including UTI, urinary tract infection [6], skin and soft tissue infection [10]; **p = 0.04 and ***p = 0.03; other comparisons are p ≥ 0.05
The ESBL-encoding gene was blaCTX-M-type in 10 of the isolates from infections (Table 3). The blaCTX-M-15 was the predominant ESBL-encoding gene. We obtained four transconjugants containing blaCTX-M-15 and two containing blaCTX-M-2, all with an ESBL phenotype and resistance to amikacin and gentamicin. The blaCTX-M-2 transconjugants were additionally resistant to sulfamethoxazole-trimethoprim. No isolate showed the presence of blaKPC or blaNDM genes.
Table 3.
Characteristics of Klebsiella pneumoniae clonal complexes observed in this study
| CC | ST (n isolates sequenced) | Caseb (n isolates)/outcome | ESBL type (n isolates) | Hosp. | Other places CC found |
|---|---|---|---|---|---|
| 45 | 45 (6) | BSI (5)/1 death related to BSI | CTXM-15 (3) | C | France, Netherlands, Spain, Japan, USA, India, UK, China, Lebanon, Poland, Italy, Ireland, Russia |
| ESBL negative (2) | C, A | ||||
| Rectal swab (1) | ESBL +, UD (1) | B | |||
| 1041 | 874/1041 SLV (4) | BSI (6)/2 deaths related to BSI, one death unrelated | CTXM-15 (4) | C | China, Japan, USA, Taiwan, Italy, France, Hungary |
| 1041a (2) | CTXM-15 (2) | ||||
| 17 | 1121a/20 SLV (1) | BSI (1)/1 death related to BSI | ESBL negative (2) | B | Germany, Netherlands, Spain, Canada, India, Vietnam, Mexico, Indonesia, Malaysia, Singapore, UK, USA, Argentina, Russia, Ireland |
| 20 (1) | BSI (1)/discharge | A | |||
| 1119a (2) | Rectal swab (2) | ESBL +, UD (2) | A | ||
| 36 | 433/36 SLV (1) | BSI (1)/1 death related to BSI | ESBL negative (2) | B | Greece, Vietnam, Australia, Spain, France, USA, Ireland, Russia |
| 36 (1) | BSI (1)/1 death related to BSI | ||||
| 200 (1) | UTI (1)/discharge | ESBL negative (1) | |||
| 611 | 483 (3) | Rectal swab (1) | ESBL +, UD (1) | B | Spain, USA, China |
| Rectal swab (1) | CTX-M-8-group (1) | ||||
| Urine (1)/discharge | ESBL negative (1) | ||||
| 14 | 14 (2) | Rectal swab (1) | ESBL +, UD (1) | B | Italy, Curacao, USA, Netherlands, France, Spain, India, Portugal, Colombia, Argentina, Australia, Vietnam, France, Norway, China, Ireland, Russia |
| BSI (1)/1 death related to BSI | ESBL negative (1) | A | |||
| 29 | 29 (1) | BSI (1)/discharge | ESBL negative (1) | B | Spain, France, Greece, Japan, Australia, United Arab Emirates, USA, Senegal, Italy, China, India, Ireland, Russia |
| 1039a/29 SLV (1) | Rectal swab (1) | ESBL +, UD (1) | |||
| 37 | 37 (2) | Rectal swab (1) | ESBL negative (2) | B | USA, Italy, Germany, South Korea, Netherlands, Senegal, Spain, Vietnam, Japan, Mexico, UK, China, Argentina, Norway, Ireland |
| Rectal swab (1) | A | ||||
| 1040 | 1040a (1) | UTI (1)/1 death related | ESBL negative (2) | A | China, Italy, Cuba, Colombia |
| 1036a/1040 SLV (1) | Tracheal aspirate (1) | ||||
| 23 | 1038a (1) | BSI (1)/discharge | ESBL negative (1) | A | Belgium, Netherlands, Spain, France, Taiwan, Vietnam, Madagascar, Australia, Singapore, Laos |
| 76 | 76 (1) | BSI (1)/discharge | ESBL negative (1) | A | USA, Netherlands, Vietnam, UK, Greece, Taiwan, Thailand, Spain, China |
| 152 | 152 (1) | BSI (1)/1 death related to BSI | ESBL negative (1) | A | Netherlands, China, Greece, Taiwan, USA, Germany, Poland, Laos, Kuwait, Spain, Algeria, Canada |
| 198 | 198 (1) | Rectal swab (1) | CTXM-2 (1) | A | Spain, China, USA, Ireland |
| 405 | 405(1) | BSI (1)/1 death related to BSI | CTX-M-8-group (1) | C | Italy, Spain, Venezuela, Norway |
| 541 | 584 (1) | Pylorus secretion (1) | ESBL negative (1) | A | Slovenia, Israel, UK, Netherlands, Taiwan, USA, Spain, Australia |
| 1723 | 334 (1) | Rectal swab (1) | ESBL negative (1) | A | Vietnam, China, USA |
| 1732 | 1035a (1) | BSI (1)/discharge | ESBL negative (1) | C | France, USA, Spain, Vietnam, China, UK, Thailand |
| Sn | 971 (1) | BSI (1)/discharge | ESBL negative (1) | A | Spain |
| Sn | 1037a (1) | BSI (1)/1 death unrelated to BSI | ESBL negative (1) | A | None |
aST first described in this study; Sn, singleton; SLV, single locus variant. bCase of infection or colonization by K. pneumoniae ESBL +, ESBL producer; UD, undetermined
Typing by ERIC-PCR was feasible for 78 of the 79 isolates, which yielded 51 distinct types. We selected for MLST 43 isolates that caused at least one infection and from isolates obtained from colonization, we choose one of each ERIC-type detected in more than one patient. Among the 26 ST detected, nine were new: ST 1035, 1036, 1037, 1038, 1039, 1040, 1119, 1120, and 1121; and three had new alleles: one for pgi (allele 100) and two for infB (alleles 79 and 80). Isolates belonging to ST14, ST45, and ST483 caused both infection and colonization. To designate similarity among ST that differed by one or two alleles forming a clonal complex (CC), we performed a minimum spanning tree analysis (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). Strains belonging to CC45 and CC1041 comprised 12 (44%) of the 27 K. pneumoniae infection isolates. CC45 was found in the 3 NICUs, in both periods of the study. Characteristics of CC and patients infected by them are shown in Table 3.
Discussion
To our knowledge, this is the first multicenter study to provide data on the genetic diversity of Klebsiella sp. isolates causing infections among neonates admitted to a NICU in a Brazilian city. Studies that reported on the genetic diversity of Klebsiella spp. were generally performed on selected samples of MDR isolates, not providing data on the overall distribution of genotypes in the studied population. In addition, few reports specifically addressed this issue among newborns admitted to NICU. It is conceivable the unique pattern of host-pathogen interaction that results from the immatureness of the immune response in this population might influence the genetic diversity of Klebsiella spp. and other pathogens. The large number of Klebsiella infections in NICU of Rio de Janeiro and the prospective study design enabled us to determine the incidence (9.5/1000 admitted patients). Such information is often unreported in other regions of the world, which makes difficult to perform geographic and institutional comparisons of the magnitude of this problem. Data from Latin America show that the incidence of Gram-negative infections as a group ranged from 35 to 580/1000 patients admitted to NICU from 1991 to 2010, most of them caused by Klebsiella (21%, in Peru, 52% in Brazil) [5]. More recent data from the Brazilian National Program for the Prevention and Control of Health-Care Related Infections provide evidence that the burden associated with infections among newborns admitted to NICU remains high, albeit with a heterogeneous distribution between different regions of the country [26]. Pooled national data from this registry showed that, in 2016, K. pneumoniae was the second most frequent pathogen causing CVC-related BSI.
In the present study that focused on newborns, a diversity of K. pneumoniae genotypes was observed (26 ST among 38 isolates). These 26 ST belonged to 17 CC, 12 (71%) of which have been described in at least three continents [24]. Therefore, neonatal K. pneumoniae infections in NICU in Rio de Janeiro appeared to be mostly polyclonal, caused by strains related to globally widespread lineages, as well as new STs, most of which were ESBL non-producers. This observation suggests there were multiple point sources of these infections. However, we also noticed that, of 27 K. pneumoniae infections with isolates stored for further studies, 12 (37%) were caused by just two CC (CC45 and CC1041), within which most MDR were clustered. In a study performed in a single-center pediatric North American hospital, albeit unrestricted to newborn population, with a similar epidemiological design, 71 ST were found among 92 K. pneumoniae isolates [27]. Most ST were represented by a single isolate, three of them the same we found in Rio de Janeiro NICU (ST45, ST20, ST37), but none of these resistant to cephalosporins. In North America and many other regions of the world, ST belonging to CC258, a KPC-2- producer, dominates published studies that describe mostly adults with underlying medical conditions. We did not find CC258 in this study.
Evidence for clonal spread is also provided by the observation that almost all strains carrying the blaCTX-M-15 gene belonged to CC45 and CC1041 isolates recovered from the same NICU. This latter clone continued to evolve, as another surveillance study performed in 2013–2014 in Rio de Janeiro found an ST1041 isolate carrying the blaOXA-370 carbapenemase gene on a plasmid [28]. Unfortunately, the kind of hospital ward where the isolate was obtained was not described. More recently, isolates belonging to clones detected in the present work were obtained from neonates elsewhere in the world, but more resistant: ST17, ST20, ST433, ST37, and ST76 were described carrying the blaNDM-1 gene [29–32]. Very importantly, one ST37 isolate obtained in Rio de Janeiro in 2013 was already resistant to trimethoprim-sulfamethoxazole, third- and fourth-generation cephalosporins, meropenem, imipenem, and colistin [32]. However, the patient or type of ward where the isolate was obtained was not described.
Lastly, we must indicate important limitations in this study. The incidence of Klebsiella sp. infections may have been underestimated, as only newborns admitted to NICUs within 24 h of birth were included. However, this criterion was important to assure a standardized neonatal population. The relatively small sample size of K. pneumoniae isolates precluded accurate determination of the clonal distribution associated with infections. However, these isolates represent a population-based sample from NICU in three large hospitals in one city, and our main observation that most of these infections were polyclonal is supported by our data. Finally, the time elapsed since this study was carried out decreases the chance that these results could accurately reflect the current distribution of K. pneumoniae clones causing infection in newborns admitted to NICUs in Brazil. Nonetheless, the reported data provide a baseline snapshot to which later data from similar studies could be compared. Such comparisons enrich our understanding on the evolution of the population structure of these pathogens, especially after strains with extensive antimicrobial resistance emerged as a cause of infection among Brazilian NICUs.
Conclusions
During the study period, the high incidence of Klebsiella sp. infections among newborns, admitted to three different NICUs in Rio de Janeiro, Brazil, appeared to be due to a combination of frequent sporadic infections caused by multiple genotypes and small outbreaks caused by dominant multidrug-resistant clones. Further studies are warranted to determine how genotype diversity of Klebsiella spp. has evolved in the NICU over time, especially after the emergence of carbapenemase-producing strains.
Acknowledgments
We thank platform Genotyping of Pathogens and Public Health (Institut Pasteur, Paris, France) for curating MLST allele sequences and profiles and making them available at http://bigsdb.pasteur.fr.
Financial support
This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Finance Code 001, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) of Brazil and Fogarty International Program in Global Infectious Diseases (TW006563) of the National Institute of Health.
Compliance with ethical standards
The study was approved by the ethics committees of the Brazilian institutions Hospital Universitário Clementino Fraga Filho (Protocol No. 235/04) and of Secretaria Municipal de Saúde of Rio de Janeiro (Protocol No. 75/08).
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
Livia Helena Justo-da-Silva wrote this manuscript.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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