Lack of correlation between growth rate and sequence type among Cronobacter sakazakii.

Species identification and growth rates for a collection of Cronobacter strains from clinical and non-clinical sources have been previously reported. However, advancements in DNA sequencing-based identification methods now allow for more accurate identification. Here we report the sequence types (STs) for 24 strains of Cronobacter sakazakii and examine any possible correlation between sequence type and growth rate, which could influence risk through greater pathogen multiplication and reach of infectious doses during time between formula preparation and feeding. The most common clonal complexes (CCs) identified were C. sakazakii CC1 and CC4. CC1 strains belonged to ST1 (n = 8) and ST391 (n = 1), while CC4 included ST4 (n = 4), ST255 (n = 1) and ST295 (n = 1). Three strains were found to belong to CC100 and two were found to belong to ST64. The remaining STs identified were represented by single strains. CC4 strains have a slightly not significant tendency for faster growth rates at 25 °C; however, the small sample size suggests that more strains need to be analysed to determine if this is a true result. In conclusion, the growth rates of C. sakazakii strains do not appear to be strongly correlated to ST.

Virulence and Antibiotic Resistance Profiles of Cronobacter sakazakii and Enterobacter spp. Involved in the Diarrheic Hemorrhagic Outbreak in Mexico.

Cronobacter spp. are bacterial pathogens that cause neonatal meningitis, septicemia, and necrotizing enterocolitis in infants with a lethality rate of 40-80%. Powdered infant formulas (PIF) have been implicated as the main vehicles of transmission. This pathogen can also cause infection through contaminated expressed breast milk, and it has been recovered from neonatal feeding tubes of neonates not fed reconstituted PIF and milk kitchen areas. This study analyzed antibiotic resistance profiles and the tissue virulence tests of Cronobacter sakazakii and Enterobacter spp. recovered from PIF, infant fecal matter's, and milk kitchen environment involved in a diarrheic hemorrhagic outbreak in 2011 in Mexico. The strains isolated from the outbreak had similar antibiotic resistance profiles and pathogenicity irrespective of isolation site, however, C. sakazakii strains isolated from PIF showed significantly higher invasive profiles than Enterobacter spp. (p = 0.001) and 83% were resistant to more than one antibiotic. The findings of this study can be used to complement existing information to better control Cronobacter and Enterobacter spp. contamination in PIF production, prevent its transmission, and improve infant food safety.

Comparative study of Cronobacter identification according to phenotyping methods.

BACKGROUND: Microbiological criteria applied to powdered infant formula (PIF) require the absence of all Cronobacter spp. Consequently, misidentification of isolates from finished products can lead to significant financial losses for manufacturers and could increase the risk of neonatal infection. Biochemical identification of suspect isolates using commercially available test panels is recommended for use by PIF manufacturers by both the US FDA and ISO standard methods for Cronobacter species; however, phenotyping can be unreliable, particularly for a genus such as Cronobacter where the taxonomy has been subject to frequent changes. This study compared the predicted identification by commonly used phenotyping kits (API20E and ID32E) for over 240 strains of Cronobacter from diverse sources, which had been identified using DNA sequence analysis. In 2015, the databases associated with the API20E and ID32E biochemical test panels were updated, including the recognition of the Cronobacter genus. Thus, the identifications from multiple versions the databases were compared to each other and to identifications based on DNA sequencing methods. RESULTS: Using previous versions of the API20E database, 90.0 % of strains (216/240) resulted in a match for the species identification; however, version 5.0 produced matches for only 82.3 % of strains (237/288). Similarly, the update to version 4.0 in the ID32E database caused the percentage of matches to drop from 88.9 % (240/270) to 43.2 % (139/322). A smaller study showed that the Vitek GN system identified all 14 strains, belonging all seven Cronobacter species, as members of the 'C. sakazakii group,' but also attributed three strains of Franconibacter helveticus and F. pulveris to this group. In silco analysis of a PCR-based method targeting ompA predicted that amplification would only occur with Cronobacter species and this method may be a feasible alternative to biochemical phenotyping. CONCLUSIONS: These results indicate that commercially available biochemical test panels are not sufficiently reliable for speciation of Cronobacter isolates. Although DNA-sequence based methods would be the more reliable approach; however, this is not currently feasible for many food microbiology laboratories. Instead, a previously published PCR-based method targeting ompA is suggested as an alternative for identification of Cronobacter species based on in silico analysis.

Draft Genome Sequence of an Enterobacter Species Associated with Illnesses and Powdered Infant Formula.

This is the first report of the draft genome sequence of an Enterobacter species that may have been transmitted from powdered infant formula (PIF) to infants, resulting in illness. Enterobacter spp. are currently permitted in PIF, but the transmission of this strain indicates that the microbiological criteria for PIF may need revision.

Reevaluation of a Suspected Cronobacter sakazakii Outbreak in Mexico.

In 2010, two infants became ill at a hospital in Mexico. Subsequently, a range of clinical, environmental, and powdered and rehydrated infant formula isolates were identified by using a combination of phenotyping and PCR probes. The strains were clustered according to pulsed-field gel electrophoresis. The causative agent was reported as Cronobacter sakazakii, with powdered infant formula (PIF) identified as the likely source of the infections. This new study further characterized the isolates from this outbreak by using multilocus sequence typing and whole genome sequencing of selected strains. Though four PIF isolates and one hospital environmental isolate were identified as C. sakazakii sequence type 297 by multilocus sequence typing, they were isolated 6 months prior to the outbreak. Genotypic analyses of patient isolates identified them as Enterobacter hormaechei and Enterobacter spp. The pulsed-field gel electrophoresis profile of the Enterobacter spp. isolates matched those of isolates from previously unopened tins of PIF. E. hormaechei was only isolated from the two infants and not PIF. The reevaluation of this outbreak highlights the need for accurate detection and identification assays, particularly during outbreak investigations in which incorrect identifications may mislead the investigation and attribution of the source. Though the species responsible for the symptoms could not be determined, this outbreak demonstrated the possible transmission of Enterobacter spp. from PIF to infants. These are possibly the first reported cases of Enterobacter spp. infection of infants from bacterial-contaminated PIF.

Description of Siccibacter colletis sp. nov., a novel species isolated from plant material, and emended description of Siccibacter turicensis.

A re-evaluation of the taxonomic position of two strains, 1383(T) and 2249, isolated from poppy seeds and tea leaves, which had been identified as Siccibacter turicensis (formerly Cronobacter zurichensis ), was carried out. The analysis included phenotypic characterization, 16S rRNA gene sequencing, multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, fusA, glnS, gyrB and infB; 2034 bp) and ribosomal MLSA (53 loci; 22 511 bp). 16S rRNA gene sequence analysis and MLSA showed that the strains formed an independent phylogenetic lineage, with Siccibacter turicensis LMG 23730(T) as the closest neighbour. Average nucleotide identity analysis and phenotypic analysis confirmed that these strains represent a novel species, for which the name Siccibacter colletis sp. nov. is proposed. The type strain is 1383(T) ( = NCTC 14934(T) = CECT 8567(T) = LMG 28204(T)). An emended description of Siccibacter turicensis is also provided.

Detection and enumeration of four foodborne pathogens in raw commingled silo milk in the United States.

A nationwide survey was conducted to obtain qualitative and quantitative data on bacterial contamination of raw commingled silo milk intended for pasteurization. The levels of total aerobic bacteria, total coliforms, Enterobacteriaceae, Escherichia coli, and Staphylococcus aureus were determined using the TEMPO system. The prevalence rates and levels of presumptive Bacillus cereus, E. coli O157:H7, Listeria monocytogenes, and Salmonella spp. were determined in 214 samples. B. cereus was detected in 8.91% of samples, at 3.0 to 93 CFU/ml. E. coli O157:H7 was detected in 3.79 to 9.05% of samples, at <0.0055 to 1.1 CFU/ml, depending on the assay utilized. Salmonella spp. were recovered from 21.96 to 57.94% of samples, at <0.0055 to 60 CFU/ml. L. monocytogenes was detected in 50.00% of samples, at <0.0055 to 30 CFU/ml. The average log-transformed counts of total viable bacteria were slightly lower in samples containing no pathogens. No correlation was observed between the levels of organisms detected with the TEMPO system and the presence or levels of any pathogen except E. coli O157:H7. A higher average log-transformed count of total viable bacteria was observed in samples positive for this organism. The high prevalence rates of target pathogens may be attributed to a variety of factors, including detection methods, sample size, and commingling of the milk in the silo. The effects of commingling likely contributed to the high prevalence rates and low levels of target pathogens because of the inclusion of milk from multiple bulk tanks. The high prevalence rates also may be the result of analysis of larger sample volumes using more sensitive detection methods. These quantitative data could be utilized to perform more accurate risk assessments and to better estimate the appropriate level of protection for dairy products and processing technologies.

Nonribosomal peptide synthetases involved in the production of medically relevant natural products.

Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.