Prevalence and Genetic Diversity of Cronobacter Species Isolated From Four Infant Formula Production Factories in China

Members of the genus Cronobacter are an emerging group of opportunist Gram-negative pathogens. This genus was previously thought to be a single species, called Enterobacter sakazakii. Cronobacter spp. typically affect low-birth-weight neonates, causing life-threatening meningitis, sepsis and necrotizing enterocolitis. Outbreaks of disease have been associated with contaminated infant formula, although the primary environmental source remains elusive. Advanced understanding of these bacteria and better classification has been obtained by improved detection techniques and genomic analysis. Research has begun to characterize the virulence factors and pathogenic potential of Cronobacter. Investigations into sterilization techniques and protocols for minimizing the risk of contamination have been reviewed at national and international forums. In this review, we explore the clinical impact of Cronobacter neonatal and pediatric infections, discuss virulence and pathogenesis, and review prevention and treatment strategies.

Cronobacter (previously known as Enterobacter sakazakii) is a diverse bacterial genus consisting of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. universalis, C. muytjensii, C. dublinensis, and C. condimenti. In this study, we have used a multilocus sequence typing (MLST) approach employing the alleles of 7 genes (atpD, fusA, glnS, gltB, gyrB, infB, and ppsA; total length, 3,036 bp) to investigate the phylogenetic relationship of 325 Cronobacter species isolates. Strains were chosen on the basis of their species, geographic and temporal distribution, source, and clinical outcome. The earliest strain was isolated from milk powder in 1950, and the earliest clinical strain was isolated in 1953. The existence of seven species was supported by MLST. Intraspecific variation ranged from low diversity in C. sakazakii to extensive diversity within some species, such as C. muytjensii and C. dublinensis, including evidence of gene conversion between species. The predominant species from clinical sources was found to be C. sakazakii. C. sakazakii sequence type 4 (ST4) was the predominant sequence type of cerebral spinal fluid isolates from cases of meningitis.

To the Editor: To overcome various limitations of phenotyping and 16S rDNA sequence analysis of Cronobacter bacteria, we have established a comprehensive multilocus sequence typing (MLST) scheme as an open access database resource (www.pubMLST.org/cronobacter) ( 1 ). The scheme is based on 7 housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3,036 nt concatenated length) and has been used to study the diversity of the Cronobacter genus and new Cronobacter species ( 2 – 4 ). Previously, we compared the sequence type profile to severity of infection by compiling patient details, isolation site, and clinical signs and symptoms for strains isolated from around the world during 1953–2008 ( 5 ). This study revealed that most serious meningitis clinical cases caused by Cronobacter spp. in neonates during the previous 30 years in 6 countries were caused by a single sequence type (ST): C. sakazakii ST4. We were therefore interested in applying the MLST method to the Cronobacter strains associated with the highly publicized cases in the United States during December 2011 ( 6 ). The Centers for Disease Control and Prevention (CDC) sent us the Cronobacter isolates they collected during 2011 for MLST analysis (Table). Ten specimens were clinical isolates from neonates or infants. These included 2 specimens (1577, 1579) associated with Cronobacter infections in Missouri and Illinois ( 6 ). Four specimens were from opened tins of powdered infant formula (PIF), and 1 was from PIF reconstitution water. DNA sequences for all specimens are available for download and independent analysis through the open access database. Table Multilocus sequence typing of Cronobacter isolates received by the CDC during 2011* Cronobacter species NTU strain ID no. CDC patient ID no. ST Location Isolation source Comment C. sakazakii 1579 2012-05-05 4 Missouri CSF of <1 mo male, term infant; exposed to PIF Patient died 1566 2011-12-02 4 Ohio CSF of 1 mo male infant; exposed to PIF From twin of patient 2011-12-03 1567 2011-12-03 4 Ohio Feces of 1 mo male infant; exposed to PIF From twin of patient 2011-12-02; asymptomatic 1568 2011-12-04 4 Ohio Opened PIF Formula associated with 2011-12-02 and -03 1570 2011-21-01 4 Minnesota CSF of <1 mo male, term infant; exposed to PIF Brain infarction 1571 2011-21-03-01 4 Minnesota Opened PIF Formula associated with 2011-21-01 1576 2193-02 4 Michigan Tracheal secretion of <1 mo male, pre-term infant (30-wk EGA); not exposed to PIF Symptoms were not caused by Cronobacter infection. Fortified breast milk fed only after culture was obtained 1565 2011-12-01 107 Michigan CSF of <1 mo male, term infant; exposed to PIF Brain abscess; outcome unknown. Single locus variant of ST4 1572 2011-21-03-02 108 Minnesota Opened PIF Single locus variant of ST4 1577 2193-03 110 Illinois CSF of 1 mo female, term infant; exposed to PIF Triple locus variant of ST4 1578 2193-08-01 111 Illinois PIF reconstitution water Bottled water associated with 2193-03 case 1573 2011-18-05-02 8 Ohio Opened PIF Formula associated with 2011-18-01 and 2011-18-07 1574 2011-18-01 8 Ohio Feces of 4 mo female, term infant; exposed to PIF Diarrheal symptoms 1575 2011-18-07 8 Ohio Feces of ≈5 mo female, term infant; exposed to PIF Ongoing diarrhea; same patient as 2011-18-01 C. malonaticus 1569 2193-01 112 Wisconsin Blood of <1 mo male pre-term infant (32 week EGA); exposed to PIF Clinical meningitis; patient died *NTU, Nottingham Trent University; ID, identification; CDC, Centers for Disease Control and Prevention; ST, sequence type; CSF, cerebrospinal fluid; PIF, powdered infant formula; EGA, estimated gestational age. Most (14/15) specimens were C. sakazakii; 1 was C. malonaticus. This predominance of C. sakazakii isolates matches reports of cases and outbreak studies ( 7 ). The C. sakazakii isolates were in 6 of 55 STs defined for C. sakazakii ( 4 ). However, there was an uneven distribution according to clinical records: all 5 cerebrospinal fluid (CSF) isolates were either ST4 or within the ST4 complex (clonal group where strains are identical in 4 or more loci). This group included strains from cases during December in Illinois (specimen 1577) and in Lebanon, Missouri (specimen 1579). Specimen 1577 (ST110), isolated from CSF, is a triple-loci variant of ST4, distinguished by 5/3036 nt: atpD (1/390nt), gltB (2/507nt), and gyrB (2/402nt). Specimen 1578 (ST111), isolated from the PIF reconstitution water associated with the case reported in Illinois, is distinguishable from ST4 in 4/7 loci: fusA (5/438nt), glnS (1/363), infB (4/441), and ppsA (19/495). The 2 Illinois strains, 1577 and 1578 (ST110 and ST111), differed from each other at all loci, in total, 35/3,036 nt difference. Such sequence-based relationship analysis of isolates is not possible by using pulsed-field gel electrophoresis (PFGE). PFGE and MLST analyze the bacterial DNA content differently, and there are no XbaI sites (the endonuclease most commonly used with PFGE of Enterobacteriaceae) within the 7 MLST loci. C. sakazakii ST4 strains were also found in feces (specimen 1567), opened PIF (specimen 1571), and tracheal samples (specimen 1576) (Figure). In addition, 2 single-loci ST4 variants were found; CSF specimen 1565 differed from the ST4 profile in the fusA loci by 6/438 nt, and specimen1572 from an opened tin of PIF differed in the fusA loci by 5/438 nt. These 2 strains differ from each other minimally, by 1 nt of 3,036 (concatenated length) in the fusA loci position 378 (A:T). Figure Maximum-likelihood tree based on the concatenated sequences (3,036 bp) of the 7 multilocus sequence type (ST) loci for Cronobacter isolates. The tree was drawn to scale by using MEGA5 (www.megasoftware.net), with 1,000 bootstrap replicates. Numbers on branches indicate percentage of bootstrap values. Scale bar indicates nucleotide substitutions per site. Several non-ST4 C. sakazakii strains were received by CDC in 2011. C. sakazakii ST8 was isolated from an opened powdered infant formula tin (specimen 1573) and 2 associated fecal samples from an infant who had diarrhea (specimens 1574 and 1575). One blood isolate (specimen 1569) was C. malonaticus ST112, found in an infant <1 month of age with meningitis who did not survive the infection. This finding is highly noteworthy because it has been proposed that C. malonaticus predominates in adult infections ( 5 ), and no fatal meningitis cases have been attributed to this species. This MLST analysis of 15 strains received by the CDC in 2011 reinforces the conclusion that CSF isolates are not evenly spread across the 7 Cronobacter species and are instead predominantly in the C. sakazakii ST4 clonal complex. Such infections in neonates are of high concern because of the risk for associated severe brain damage. As previously stated, whether this association is caused by greater neonatal exposure as a result of environmental factors or particular virulence capabilities remains uncertain ( 5 ).