Evaluation of phenotypical and genotypical methods for the identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility.

AIMS: Evaluate methods for identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility. METHODS AND RESULTS: From 270 S. maltophilia strains identified by VITEK®2, 40 were selected and submitted to MALDI TOF-MS, 16S and 23S rRNA gene analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and an antimicrobial susceptibility profile. 16S rRNA sequencing was able to identify 39 (97.5%) strains as Stenotrophomonas spp. and one (2.5%) as Luteimonas huabeiensis. MALDI TOF-MS identified 37 (92.5%) strains as S. maltophilia, and three (7.5%) were not identified. PCR targeting 23S rRNA yielded a positive result for 39 (97.5%) strains. However, after sequencing, two strains were identified as Stenotrophomonas rhizophila, showing false-positive results. The confirmed S. maltophilia strains (n = 37) showed 35 distinct ERIC-PCR profiles and exhibited sensitivity to minocycline and levofloxacin, and six (16.3%) showed intermediate resistance to sulfamethoxazole-trimethoprim. CONCLUSION: Matrix-assisted laser desorption lonization-time of flight mass spectrometry (MALDI-TOF MS) was a satisfactory methodology for the identification of S. maltophilia, but expansion of the database is necessary for the identification of other species. 16S rDNA sequencing showed low resolution for Stenotrophomonas species differentiation. PCR targeting 23S rRNA could not differentiate S. maltophilia from S. rhizophila. ERIC-PCR was shown to be a useful tool for the microbial source tracking of S. maltophilia.

Diversity and epidemiological profile of Pseudomonas aeruginosa from drinking water in Brazil genotyped using multi-locus sequence typing.

Pseudomonas aeruginosa is a Gram-negative bacillus associated with waterborne diseases. The objective of this study was to determine whether particular P. aeruginosa sequence types (STs) were associated with drinking water contamination in Brazil. This was achieved by searching the Pseudomonas PubMLST database, which contains the records for 8358 strains collected between 1938 and 2023. The majority (97.2%) had the complete 7-loci multilocus sequence typing profile and were assigned to 3486 STs. After eBURST (an algorithm used to infer patterns of evolutionary descent among clusters), 1219 groups with single-locus variant and 575 groups with double-locus variant were formed. Brazil was the South American country with the most isolates (n = 219, 58.24%), and the Simpson's index was 0.9392. Of the 219 Brazilian isolates, eight were isolated in water and identified as STs 252, 1417, 1605, 2502, 2620, 3078, and 3312. ST252, 1417, and 3078 have already been isolated from clinical cases worldwide. Furthermore, ST1605 and 2620, after the eBURST, they were grouped in the same clonal complex as STs involved in human infections. In conclusion, P. aeruginosa STs involved in human infections were found in bottled drinking water commercialized in Brazil, revealing that these types of drinking waters can be a vehicle of contamination.

Phenotypical and molecular characterization of Acinetobacter spp. isolated from a pharmaceutical facility.

Characterizing microorganisms according to different criteria is useful when investigating sources of microbiological contamination in the pharmaceutical industry. The aim of this study was to characterize 38 Acinetobacter baumannii complex strains isolated from a biopharmaceutical industry by 16S rRNA sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), multilocus sequence typing (MLST), antimicrobial susceptibility profile, biofilm formation, and sensibility to disinfectants. Thirty-three (86.9%) strains were identified by 16S rRNA gene sequencing as A. seifertii/pitti/nosocomialis/lactucae, four (10.5%) as A. baumannii, and one (2.6%) as A. vivianii/courvalini. MALDI-TOF/MS did not identify one strain, and incorrectly identified 30/37 (81.1%) strains as A. baumannii. Strains were assigned to 12 different STs, of which nine were newly defined in this study (STs 2091-2099). Twenty-six (68.4%) strains showed resistance to amikacin and gentamicin. Thirty-three (86.8%) strains were classified as moderately or strongly adherent on polystyrene. Alcohol 70%/15 min and quaternary ammonium 0.08%/20 min were not able to eliminate the biofilm formed, but sodium hypochlorite 0.1%/15 min was efficient. In conclusion, improved methods are needed to improve the identification of Acinetobacter strains in pharmaceutical industries. This organism is of particular concern as it forms recalcitrant biofilms, leading to persistence in the manufacturing environment and increased risk of product contamination.

Identification of Sutcliffiella horikoshii strains in an immunobiological pharmaceutical industry facility.

The pharmaceutical industry must comply with the requirements for good manufacturing practices to reduce inherent contamination risks in the production process. Bacillus and related genera are among the main bacterial isolated from clean areas, raw material, and products in the pharmaceutical industries, but the correct identification of these species is still a challenge. The aim of this study was to characterize by phenotyping, protein profiling, and 16S rRNA gene sequencing Sutcliffiellahorikoshii strains (n = 6) isolated from an immunobiological pharmaceutical facility, and to propose the reclassification of Bacillus tianshenii to the genus Sutcliffiella, and Sutcliffiella tianshenii sp. nov. The strains were characterized by VITEK®2, matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) using VITEK®MS, and 16S rRNA gene sequencing analysis. MALDI-TOF/MS did not identify any strains that were identified by 16S rRNA as S. horikoshii. VITEK®2 showed false-positive results, with misidentification as B. sporothermodurans (reclassified as Heyndrickxia sporothermodurans) and Geobacillus thermoleovorans. After MALDI-TOF/MS database expansion, with the creation of SuperSpectrum, the strains were correctly identified as S. horikoshii. This study is the first report of isolation of S. horikoshii strains from a pharmaceutical industry. More studies are necessary to better understand the ability of S. horikoshii to contaminate the environment and products.

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.

Outbreak of Cronobacter turicensis in European brown hares (Lepus europaeus).

This is the first report of acute deaths in five European brown hares (Lepus europaeus) attributed to mucoid and necrotizing typhlocolitis caused by genetically different Cronobacter (C.) turicensis strains in northeastern Austria. As this opportunistic pathogen is mainly known for causing disease in immunocompromised humans and neonates, this previously unrecognized potential for a spill over from a wildlife reservoir to humans warrants further attention.

Meningitis and brain abscess formation caused by Cronobacter malonaticus sequence type 440 in a full-term neonate.

Cronobacter spp. cause serious diseases, such as necrotizing enterocolitis, bacteremia, and meningitis in neonates and infants. Most Cronobacter-associated meningitis is reportedly due to C. sakazakii and the majority of infections caused by C. malonaticus occur in adults and are less severe. We report the case of meningitis and brain abscess caused by C. malonaticus Sequence Type (ST) 440 in a healthy full-term neonate. We should consider the possibility that full-term neonates may develop meningitis due to C. malonaticus and treat appropriately because its mortality rate is very high, and survivors are usually left with severe neurologic impairment. In addition, C. malonaticus ST440 may have virulence factors that cause neonatal meningitis akin to the previous report of meningitic ST307 strain.

Applicability of Enterobacteriaceae and coliforms tests as indicators for Cronobacter in milk powder factory environments.

The emergence of Cronobacter as an important potential pathogen for newborn children and its occurrence in powdered infant formulae has generated a need to develop new management practices for this food group. This includes reduction of the prevalence of Cronobacter in manufacturing environments which can be a source of Cronobacter. This study was performed to assess the suitability of qualitative and quantitative Enterobacteriaceae and coliforms indicator tests for the presence and prevalence of Cronobacter. Environmental swabs (205) from five milk powder factories were examined. The qualitative indicator tests had good sensitivity but they lacked specificity for reliable routine use. Logistic regression analyses revealed a significant relationship between the quantitative indicator tests and Cronobacter prevalence, where the Enterobacteriaceae count was a slightly stronger predictor for Cronobacter than the coliforms count. The optimum test sensitivity (81%) and specificity (66%) was obtained when the indicator count thresholds were set at ≥1 cfu/cm2. However, since 11% of samples were Cronobacter positive when counts of Enterobacteriaceae and coliforms were less than 1 cfu/cm2, specific testing for Cronobacter is advised in addition to Enterobacteriaceae testing to minimise risk of transfer of Cronobacter from the factory environment into powdered infant formulae products.

Invited review: Stress resistance of Cronobacter spp. affecting control of its growth during food production.

Members of the Cronobacter genus include food-borne pathogens that can cause infections in infants, with a mortality rate as high as 40 to 80%. The high fatality rate of Cronobacter and its isolation from numerous types of food, especially from powdered infant formula, demonstrate the serious nature of this organism. The source tracking of Cronobacter spp. and the analysis of high-frequency species from different sources are helpful for a more targeted control. Furthermore, the persistence during food processing and storage may be attributed to strong resistance of Cronobacter spp. to environment stresses such as heat, pH, and desiccation. There are many factors that support the survival of Cronobacter spp. in harsh environments, such as some genes, regulatory systems, and biofilms. Advanced detection technology is helpful for the strict monitoring of Cronobacter spp. In addition to the traditional heat treatment, many new control techniques have been developed, and the ability to control Cronobacter spp. has been demonstrated. The control of this bacteria is required not only during manufacture, but also through the selection of packaging methods to reduce postprocessing contamination. At the same time, the effect of inactivation methods on product quality and safety must be considered. This review considers the advances in our understanding of environmental stress response in Cronobacter spp. with special emphasis on its implications in food processing.

Microbiological quality of kitchens sponges used in university student dormitories.

BACKGROUND: Kitchen sponges are a major source of cross-contamination as they can transfer foodborne pathogens, infectious agents and spoilage causing microorganisms to food contact surfaces. Several studies have revealed that university students adopt poor practices regarding food safety, hygiene, and the handling of kitchen cleaning equipment. METHODS: A total of fifty kitchen sponges were collected along with a questionnaire addressing social demographics and kitchen sponge usage by students living at the University of Sharjah dormitories. The effect of storage (3 and 10 days) on the microbial population of kitchen sponges at room temperature (21 °C) was assessed. Enterobacteriaceae isolated from sponges were identified and their antibiotic resistance determined. RESULTS: Student responses revealed that kitchen sponges used to clean food contact surfaces were also used to clean the oven (32%), sink (26%), refrigerator (10%), and to clean spills on the floor (4%). Kitchen sponges contained high counts of mesophilic aerobic bacteria (7.9 log10/cm3), coliform (7.2 log10/cm3), Enterobacteriaceae (7.3 log10/cm3) and yeasts and molds (7.0 log10/cm3). After storage of the sponges at room temperature (21 °C) for 3 and 10 days, the number of mesophilic aerobic bacteria, coliform, Enterobacteriaceae and yeasts and molds decreased by 0.4 and 1.3 log10/cm3, 0.7 and 1.4 log10/cm3, 0.4 and 1.1 log10/cm3, and 0.6 and 1.3 log10/cm3, respectively. The most frequently isolated Enterobacteriaceae were Enterobacter cloacae (56%) and Klebsiella oxytoca (16%). All E. cloacae isolates were resistant to amoxicillin, cefalotin, cefoxitin and cefuroxime axetil. CONCLUSIONS: This study showed that students living in dormitories lacked good hygienic practices and were at increased risk of food poisoning. Kitchen sponges were highly contaminated with potentially pathogenic bacteria which could be transferred from the general kitchen environment to food contact surfaces and consequently lead to food contamination.

Multicenter Study of Cronobacter sakazakii Infections in Humans, Europe, 2017.

Cronobacter sakazakii has been documented as a cause of life-threating infections, predominantly in neonates. We conducted a multicenter study to assess the occurrence of C. sakazakii across Europe and the extent of clonality for outbreak detection. National coordinators representing 24 countries in Europe were requested to submit all human C. sakazakii isolates collected during 2017 to a study center in Austria. Testing at the center included species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, subtyping by whole-genome sequencing (WGS), and determination of antimicrobial resistance. Eleven countries sent 77 isolates, including 36 isolates from 2017 and 41 historical isolates. Fifty-nine isolates were confirmed as C. sakazakii by WGS, highlighting the challenge of correctly identifying Cronobacter spp. WGS-based typing revealed high strain diversity, indicating absence of multinational outbreaks in 2017, but identified 4 previously unpublished historical outbreaks. WGS is the recommended method for accurate identification, typing, and detection of this pathogen.

Occurrence of virulence factors in Cronobacter sakazakii and Cronobacter malonaticus originated from clinical samples.

BACKGROUND: Cronobacter spp. are Gram-negative, facultative-anaerobic, non-spore forming, enteric coliform bacteria, which belongs to the Enterobacteriaceae family. Cronobacter spp. are opportunistic pathogens that have brought rare but life-threatening infections such as meningitis, necrotizing enterocolitis and bloodstream infections in neonates and infants. Information on the diversity, pathogenicity and virulence of Cronobacter species obtained from various sources is still relatively scarce and fragmentary. The aim of this study was to examine and analyse different pathogenicity and virulence factors among C. sakazakii and C. malonaticus strains isolated from clinical samples. METHODS: The thirty-six clinical Cronobacter strains have been used in this study. This bacterial collection consists of 25 strains of C. sakazakii and 11 strains of C. malonaticus, isolated from different clinical materials. Seven genes (ompA, inv, sip, aut, hly, fliC, cpa) were amplified by PCR. Moreover, the motility and the ability of these strains to adhere and invade human colorectal adenocarcinoma (HT-29) and mouse neuroblastoma (N1E-115) cell lines were investigated. RESULTS: Our results showed that all tested strains were able to adhere to both used cell lines, HT-29 and N1E-115 cells. The invasion assay showed that 66.7% (24/36) of isolates were able to invade N1-E115 cells while 83% (30/36) of isolates were able to invade HT-29 cells. On the average, 68% of the C. sakazakii strains exhibited seven virulence factors and only 18% in C. malonaticus. All strains amplified ompA and fliC genes. The other genes were detected as follow: sip 97% (35/36), hlyA 92% (33/36), aut 94% (34/36), cpa 67% (24/36), and inv 69% (25/36). CONCLUSIONS: C. sakazakii and C malonaticus strains demonstrate the diversity of the virulence factors present among these pathogens. It is necessary to permanently monitor the hospital environment to appropriately treat and resolve cases associated with disease. Furthermore, in-depth knowledge is needed about the source and transmission vehicles of pathogens in hospitals to adopt pertinent prevention measures.

Probiotics interaction with foodborne pathogens: a potential alternative to antibiotics and future challenges.

Antibiotics are a key tool used nowadays in health care industry to fight against bacterial infections; however, repeated antibiotic use or misuses, have led to bacterial resistance, causing significant threats for many people with common bacterial infections. The use of probiotics to enhance gastrointestinal health has been proposed for many years. In recent years, there has been an increasing interest in the use of probiotic bacteria as alternatives for antibiotics for preventing or treating various intestinal infections. Several important underlying mechanisms responsible for the antagonistic effects of probiotics on different microorganisms include: (1) competitive exclusion for adhesion sites and nutritional sources; (2) secretion of antimicrobial substances; (3) enhancement of intestinal barrier function; and (4) immunomodulation. However, their mode of action is not very well understood and therefore a clearer understanding of these mechanisms is necessitated. This will enable appropriate probiotic strains to be selected for particular applications and may reveal new probiotic functions. The goal of this review was to highlight some studies from literature describing the probiotic interaction with several major foodborne pathogens, as well as explore the mechanisms for such probiotic-pathogen interaction. The review will conclude by presenting future perspective and challenges of probiotic application in food products.

Characterization of the pathogenicity of clinical Cronobacter malonaticus strains based on the tissue culture investigations.

Cronobacter malonaticus is a member of the genus Cronobacter which is considered an opportunistic pathogen. The significance of C. malonaticus has recently increased since it was documented to be involved in several serious neonatal infections. However, the virulence factors of C. malonaticus including their ability to adhere, invade and overcome host barriers have not been studied before. Unlike previous Cronobacter research, this study is mainly focused on C. malonaticus and is aimed to investigate its virulence characteristics that enable this species to cause adult and neonatal infections. Altogether, 20 strains were included in this study (19 clinical and one environmental strain). Our data showed that the clinical C. malonaticus has an ability to adhere and invade Caco-2, HBMEC, A549 and T24 cell lines. Moreover, the result showed that certain strains of C. malonaticus (including 1827 and 2018) were able to persist well in macrophages. However, ST7 strains 1827 and 2018 proved to be the most invasive strains among all used strains. The CDC strain 1569 (ST307) which was isolated from the blood of a fatal neonatal case showed also significant results in this study as it was able to invade all used human cells and survive and replicate within microphages. Finally, the findings of this study confirm the potential ability of C. malonaticus to cause serious infections in neonates or adults such as necrotising enterocolitis, meningitis, bacteraemia, pneumonia and urinary tract infection.

Species identification and molecular characterization of Cronobacter spp. isolated from food imported over nine years into Beijing, China.

Cronobacter spp. are associated with serious infections in neonates with the clinical presentations of necrotizing enterocolitis, bacteraemia and meningitis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify 203 Cronobacter isolates from imported food during 2006-2015 with an optimized in-house database. The isolates were predominantly C. sakazakii (88.18%), followed by C. malonaticus (8.37%), C. muytjensii (1.48%), C. turicensis (0.99%) and C. dublinensis (0.99%). The result was totally consistent with that of fusA allele sequencing. 12.32% (25/203) of isolates gave inconsistent spectra following separate protein extractions. Sixty C. sakazakii isolates and 24 isolates from the other four species were chosen for multi-locus sequence type analyses (MLST) and PCR-serotyping. Thirty-one sequence types were identified. The common sequence types were ST1 (19/60) and ST4 (13/60) for C. sakazakii and ST7 (12/17) for C. malonaticus. The primary serotypes were Csak O:1 (30/60), Csak O:2 (25/60) and Cmal O:2 (16/17) for C. sakazakii and C. malonaticus isolates, respectively. In conclusion, appropriate in-house database could make MALDI-TOF MS method identifying Cronobacter spp. isolates to the species level. But the spectra data were not sufficiently consistent for subtyping, unlike MLST. The Cronobacter spp. isolates have a high diversity including recognized pathovars.

Fatal Cronobacter sakazakii Sequence Type 494 Meningitis in a Newborn, Brazil.

We describe a case of infection with Cronobacter sakazakii sequence type 494 causing bacteremia and meningitis in a hospitalized late premature infant in Brazil. We conducted microbiological analyses on samples of powdered infant formula from the same batch as formula ingested by the infant but could not identify the source of contamination.

Characterisation of Cronobacter strains isolated from hospitalised adult patients.

Bacteria belonging to the genus Cronobacter are opportunistic pathogens known for causing rare but serious infections in neonates, including meningitis, necrotising enterocolitis and sepsis. Cronobacter infections occur also in adult populations, however, they generally have milder manifestations and their prevalence is uncertain. In this study, the presence of Cronobacter strains from adult patients in the University Hospital in Bratislava was investigated and overall 18 confirmed isolates from 321 patients (5.3%) were recovered. No Cronobacter positive sample was detected in 215 sputum samples from outpatients. The highest occurrence of Cronobacter strains was observed from stroke patients and this may be associated with an abnormal swallowing ability. The isolated strains belonged to the species Cronobacter sakazakii and Cronobacter malonaticus. In silico genotyping (MLST, CRISPR-cas array profiling) of whole genome sequences assigned the strains to three different MLST clones. The majority (12/18) of the isolated strains were sequence type ST513 or single locus variants ST514 and ST515, thereby being members of C. sakazakii pathovar clonal complex CC4. However, according to core genome MLST analysis the ST513-ST515 strains created a unique cluster substantially different from other CC4 strains. The isolated strains were susceptible to 18 tested antibiotics. All strains possess a genomic island encoding for increased thermal tolerance. As Cronobacter strains are frequently present in dried foods of plant origin, spread of a specific clone within a hospital may be caused by food transmission and may be facilitated by its tolerance to environmental stresses such as desiccation and temperature.

Microbial community structure and distribution in the air of a powdered infant formula factory based on cultivation and high-throughput sequence methods.

The air in a powdered infant formula (PIF) factory is a potential transfer medium for microorganisms. In this study, air samples from 6 main processing areas, almost covering the whole PIF processing line and 1 outdoor location, were collected from a PIF manufacturing plant during the winter and summer periods. A cultivation-based and an Illumina (San Diego, CA) high-throughput 16S rRNA sequencing method was used to investigate the community structures and distributions of bacteria in the air. High microbial diversity (25 genera, 56 species), with a dominant community including Staphylococcus, Bacillus, Acinetobacter, and Kocuria, was found by the cultivation-based method. Moreover, 104 genera were obtained from all samples by high-throughput sequencing methods. Lactococcus (32.3%), Bacillus (29.6%), and Staphylococcus (14.0%) were the preponderant genera. The indices from high-throughput sequencing results indicated that the bacterial community of the air samples was highly diverse. Significant differences in the diversity and distribution at 6 sampling locations were revealed using the 2 methods. In particular, the packaging process contained the highest proportion (39.4%) of isolated strains. The highest diversity in bacterial community structure was found in the outdoor location. More bacterial isolates and higher community diversity were observed in the summer samples compared with the winter samples. In addition, some pathogens, such as Acinetobacter baumannii, Bacillus cereus, and Staphylococcus cohnii, were mainly found in the large bag filling process, can filling, and packaging process areas. The present study provides greater insight into the microbial community and identifies potential sources of air contamination in PIF production environments and can serve as a guide to reduce the risk of microbial contamination in the production of PIF.

Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar.

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.