The speciation and genotyping of Cronobacter isolates from hospitalised patients

A total of 51 Cronobacter strains were characterised by several phenotyping and genotyping methods. Presumptive identification using ID 32E phenotyping identified 49 isolates as Enterobacter sakazakii, one strain (1838) as Pantoea spp. and the remaining strain (1841) as E. cloacae. Since the bioMérieux ID 32E online database does not recognise the Cronobacter genus, the strains could not be further identified using this method.

Using the fusA sequence analysis and comparison with the Cronobacter PubMLST database identified the 51 strains as primarily C. sakazakii (33/51), followed by C. malonaticus (17/51) and one C. muytjensii strain. The strains were then further genotyped using the 7-loci MLST scheme. This supported the species identification-based fusA sequence analysis, and further subtyped the isolates (Table 2). The C. sakazakii strains were from two sequence types; ST4 (32/51, 63 %) and ST64 (1/51, 2 %). All the C. malonaticus strains were ST7 (17/51, 33 %) and the single C. muytjensii isolate was ST28 (2 %).

The identification of strains using rpoB sequence analysis [18] and comparison with rpoB sequences in the Cronobacter PubMLST database agreed with species designation using fusA allele sequence analysis (Table 2). There were four different rpoB profiles, 1, 18, 35 and 36, which correlated with their 7-loci sequences types. All C. sakazakii ST4 and ST64 strains were rpoB profiles 1 and 35, respectively. The C. malonaticus ST7 strains were rpoB profile 18 and C. muytjensii ST28 was rpoB profile 36. See Table 2 for more information.

Comparison with serotyping profiling showed a strong correlation between some sequence types and serotypes. O-serotype C. sakazakii O:2 corresponded with C. sakazakii ST4. The association was not exclusive however, as C. sakazakii ST64 (strain 1995) was also serotype C. sakazakii O:2. In addition, the serotype of all (n = 17) C. malonaticus ST7 strains corresponded with the two designated serotypes C. malonaticus O:2 and C. sakazakii O:6 according to the schemes of Jarvis et al. and Sun et al., respectively [16, 17]. Based on fusA speciation, C. malonaticus O:2 was given as the serotype for these strains (Table 3). No serotype could be determined for the C. muytjensii strain as no PCR products were obtained with either PCR serotyping scheme.

PFGE was used to ascertain whether the strains in each sequence type (i.e. C. sakazakii ST4 and C. malonaticus ST7) could be further distinguished and whether this could be used to profile the strains from the two hospitals. Using the restriction enzyme XbaI, C. sakazakii strains gave 12 to 17 DNA fragments per strain, whereas C. malonaticus strains gave 8 to 10 bands (Fig. 1). Comparable numbers of fragments were obtained using SpeI: 14 to 17 bands for C. sakazakii strains and 14 to 16 bands for C. malonaticus strains. The XbaI restriction enzyme separated the collection into 16 pulsotypes: ten for C. sakazakii, five for C. malonaticus and one for C. muytjensii, while the SpeI restriction enzyme divided the collection into 14 pulsotypes: eight for C. sakazakii, five for C. malonaticus and one for C. muytjensii. Combining the PFGE profiles generated with the restriction enzymes XbaI and SpeI grouped the 51 strains into a total of 15 pulsotypes: nine for C. sakazakii, five for C. malonaticus and one for C. muytjensii. Strains of the same sequence type from different hospital departments were distinguishable by PFGE and are considered in more detail below.

The isolates from Olomouc hospital formed four distinguishable C. malonaticus pulsotypes (PT2 to 5) and one C. muytjensii pulsotype (PT6), which were recovered from different age groups of patients from four hospital departments. PT2 was one C. malonaticus ST7 strain (1827) isolated in the Internal Medicine Department from the intravenous cannula of a 76-year-old patient in 2007. PT3 was composed of three C. malonaticus ST7 strains (1834, 1835, 1833), two of which were isolated from the Paediatric Department and one was from the Clinical and Molecular Pathology Department. The three PT3 strains had been isolated over a 2-year period from throat and stool samples of patients under 16 years of age. The six isolates in PT4 were all C. malonaticus ST7 strains. Five had been isolated from the Paediatric Department over a 10-month period from throat swabs and one from a stool sample. The patient ages ranged from 3 to 17 years old. The majority (4/5) of PT5 strains were isolated from the throat and one from nose from the same Paediatric Department. These strains were also C. malonaticus ST7 and had been collected over a period of 2 years. The patient ages ranged from 2 months to 3 years. C. muytjensii ST28 strain 1838 was in a unique pulsotype (PT6). This strain was isolated in 2012 at the Anaesthesiology and Intensive Care Unit, from the sputum of a 63-year-old patient.

The isolates from Prostějov hospital were recovered from seven departments and were clustered in ten distinguishable Cronobacter pulsotypes (Table 3). PT1 was the only C. malonaticus pulsotype (strains 1914 and 2018). These were both C. malonaticus ST7 strains which were isolated from patients’ sputum at the Infectious Disease Department. The collection was over a 7-month period, and the patients were 69 and 72 years in age. All the remaining isolates were strains of C. sakazakii, which formed nine pulsotypes (PT7 to 15). Eight of these pulsotypes (PT7 to 14) were composed of 32 strains of C. sakazakii ST4. PT15 was composed of one C. sakazakii ST64 strain (1995). Most of the 15 C. sakazakii ST4 strains in PT12 were isolated from sputum except strains 1837 and 2010, which were isolated from a wound swab and throat swab, respectively. This pulsotype was collected over period of about 1 year and the patients ages ranged from 27 to 87 years. In PT12, 12 isolates were collected from the Internal Medicine Department, two from the Anaesthesiology and Intensive Care Unit and one from the Surgery Department. PT13 and PT14 each contained single C. sakazakii ST4 strains; 1839 and 1836, respectively. PT15 contained a single C. sakazakii ST64 strain (1995). These strains were isolated from a percutaneous endoscopic gastrostomy smear ICU, wound surgery and the sputum of an outpatient, respectively. The isolations were over a 7-month period and the patient ages ranged from 50 to 73 years. PT7 consisted of seven C. sakazakii ST4; strains 1840, 1916 and 2002 were isolated from sputum, strain 2000 from rectal swab, strain 2001 from oral cavity swab, strain 2009 from tongue swab and strain 2011 from section catheter. Six of the isolates were collected from the Internal Medicine department, and strain 1840 was isolated from an Intensive Care Unit patient. The collection was over a 7-month period and all patients were over 68 years of age. PT8, 9, 10 and 11 consisted of eight C. sakazakii ST4 strains. All these strains except one (1997) were isolated from sputum at the Internal Medicine Department, whereas strain 1997 was collected from the Intensive Care Unit. The PT8 strain was isolated in 2013 from a 91-year-old patient. PT9 was isolated in 2013 from a 70-year-old patient. PT10 was collected over a roughly 8-month period and the patient ages were between 49 and 70 years old. The two strains in PT11 were collected in 2012 and 2013 and the mean patient age was 67 years (Table 4).

goeBURST analysis showed the range of patient ages and sources with Cronobacter species (Fig. 2). C. sakazakii ST4 strains were predominantly sputum samples from adults >70 years in age, whereas C. malonaticus ST7 were from throat swabs of children <6 years old.