Séamus Fanning

Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov.

[Enterobacter] sakazakii is an opportunistic pathogen that can cause infections in neonates. This study further clarifies the taxonomy of isolates described as [E.] sakazakii and completes the formal description of the proposed reclassification of these organisms as novel species and subspecies within a proposed novel genus, Cronobacter gen. nov. [E.] sakazakii was first defined in 1980, however recent polyphasic taxonomic analysis has determined that this group of organisms consists of several genomospecies. In this study, the phenotypic descriptions of the proposed novel species are expanded using Biotype 100 and Biolog Phenotype MicroArray data. Further DNA-DNA hybridization experiments showed that malonate-positive strains within the [E.] sakazakii genomospecies represent a distinct species, not a subspecies. DNA-DNA hybridizations also determined that phenotypically different strains within the proposed species, Cronobacter dublinensis sp. nov., belong to the same species and can be considered as novel subspecies. Based on these analyses, the following alternative classifications are proposed: Cronobacter sakazakii gen. nov., comb. nov. [type strain ATCC 29544(T) (=NCTC 11467(T))]; Cronobacter malonaticus sp. nov. [type strain CDC 1058-77(T) (=LMG 23826(T)=DSM 18702(T))]; Cronobacter turicensis sp. nov. [type strain z3032(T) (=LMG 23827(T)=DSM 18703(T))]; Cronobacter muytjensii sp. nov. [type strain ATCC 51329(T) (=CIP 103581(T))]; Cronobacter dublinensis sp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. dublinensis subsp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. lausannensis subsp. nov. [type strain E515(T) (=LMG 23824=DSM 18706(T))], and Cronobacter dublinensis subsp. lactaridi subsp. nov. [type strain E464(T) (=LMG 23825(T)=DSM 18707(T))].

Enterobacter sakazakii: An Emerging Pathogen in Powdered Infant Formula

Enterobacter sakazakii represents a significant risk to the health of neonates. This bacterium is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. Infants aged <28 days are considered to be most at risk. Feeding with powdered infant formula (PIF) has been epidemiologically implicated in several clinical cases. Infants should be exclusively breast-fed for the first 6 months of life, and those who are not should be provided with a suitable breast-milk substitute. PIF is not a sterile product; to reduce the risk of infection, the reconstitution of powdered formula should be undertaken by caregivers using good hygienic measures and in accordance with the product manufacturers food safety guidelines.

Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen.

Cronobacter spp. (Enterobacter sakazakii) are a recently described genus that is comprised of six genomospecies. The classification of these organisms was revised based on a detailed polyphasic taxonomic study. Cronobacter spp. are regarded as ubiquitous organisms having been isolated from a wide variety of foods. These bacteria are opportunistic pathogens and are linked with life-threatening infections in neonates. Clinical symptoms of Cronobacter infection include necrotizing enterocolitis, bacteremia, and meningitis, with case fatality rates of 50-80% being reported. Contaminated powdered infant formula has been epidemiologically linked with infections. Recently, infections among immunocompromised adults, mainly the elderly, have also been reported. A high tolerance to osmotic stress and elevated temperatures contribute to the survival of Cronobacter spp. in dried foods such as powdered infant formula. Controlling the organism in the production environment, thereby reducing dissemination, necessitates the provision of suitable diagnostic tools. Studies demonstrated that a high degree of variability exists amongst the phenotypic-based methods used to identify Cronobacter spp. However, advances in molecular detection and subtyping techniques have significantly improved the identification and characterization of Cronobacter spp. The dose required to induce infection has yet to be determined. In vitro virulence studies have shown that Cronobacter spp. may survive in macrophage cells and efficiently attach to and invade epithelial cell lines. The production of exopolysaccharide may contribute to the formation of biofilm and active efflux pumps promote resistance to antimicrobial agents such as bile salts and disinfectants. A holistic approach combining techniques such as comparative genome analysis, proteomics, and in vivo challenges could help unravel the complex interactions between this pathogen and its host. These data would help identify those properties in Cronobacter spp. which enable the bacterium to survive in the production environment and infect vulnerable neonates via the food chain.

Low-water activity foods: increased concern as vehicles of foodborne pathogens

Foods and food ingredients with low water activity (a(w)) have been implicated with increased frequency in recent years as vehicles for pathogens that have caused outbreaks of illnesses. Some of these foodborne pathogens can survive for several months, even years, in low-a(w) foods and in dry food processing and preparation environments. Foodborne pathogens in low-a(w) foods often exhibit an increased tolerance to heat and other treatments that are lethal to cells in high-a(w) environments. It is virtually impossible to eliminate these pathogens in many dry foods or dry food ingredients without impairing organoleptic quality. Control measures should therefore focus on preventing contamination, which is often a much greater challenge than designing efficient control measures for high-a(w) foods. The most efficient approaches to prevent contamination are based on hygienic design, zoning, and implementation of efficient cleaning and sanitation procedures in the food processing environment. Methodologies to improve the sensitivity and speed of assays to resuscitate desiccated cells of foodborne pathogens and to detect them when present in dry foods in very low numbers should be developed. The goal should be to advance our knowledge of the behavior of foodborne pathogens in low-a(w) foods and food ingredients, with the ultimate aim of developing and implementing interventions that will reduce foodborne illness associated with this food category. Presented here are some observations on survival and persistence of foodborne pathogens in low-a(w) foods, selected outbreaks of illnesses associated with consumption of these foods, and approaches to minimize safety risks.

Development and evaluation of rpoB based PCR systems to differentiate the six proposed species within the genus Cronobacter

Although there are various PCR based methods described in the literature to detect the genus Cronobacter, none of these methods is able to differentiate the six proposed species within this genus. A species differentiation is important for epidemiological studies. Moreover, the different species show differences in sensitivity to chemical agents and antibiotics. Here we report for the first time different rpoB based PCR systems which enable the identification to species level of strains previously confirmed to belong to the genus Cronobacter. Different primer pairs based on the rpoB sequences of the six Cronobacter species type strains were designed. Thereafter, 57 target and non-target strains, previously described in the Cronobacter taxonomy paper, were included for the specificity evaluation. C. turicensis, C. muytjensii, C. dublinensis and C. genomospecies 1 can be reliably identified by the proposed single primer pairs. Only target strains showed a correctly sized amplification product, whereas no amplification product was obtained for all non-target strains used in this study (100% specificity). However, as the rpoB gene sequences of C. sakazakii and C. malonaticus are closely related, a two step procedure is necessary. We therefore recommend a two-step procedure in which the primer pairs Cmalf/Cmalr are used in a follow up PCR on strains that are found to be positive in the amplification with the C. sakazakii specific primers Csakf/Csakr.

Emergence and control of Fluoroquinolone Resistant Toxin A Negative Toxin B positive Clostridium difficile

BACKGROUND Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Between August 2003 and January 2004, we experienced an increase in the incidence of C. difficile-associated disease. We describe the investigation into and management of the outbreak in this article. METHODS A total of 73 consecutive patients with nosocomial C. difficile-associated diarrhea were identified. C. difficile isolates were characterized using toxin-specific enzyme immunoassays, a tissue-culture fibroblast cytotoxicity assay, polymerase chain reaction (PCR), and antimicrobial susceptibility tests. Rates of recurrence and of C. difficile colitis were recorded. Changes in antibiotic use and infection control policies were documented. RESULTS The incidence of C. difficile-associated diarrhea peaked at 21 cases per 1,000 patient admissions. Of the C. difficile isolates recovered, 85 (95%) were identical toxin A-negative and toxin B-positive strains, corresponding to toxinotype VIII and PCR ribotype 017. All clonal isolates were resistant to multiple antibiotics, including ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin (minimum inhibitory concentrations [MICs] of greater than 32 micro g/mL) and erythromycin, clarithromycin, and clindamycin (MICs of greater than 256 micro g/mL). Recurrent C. difficile-associated disease occurred in 26 (36%) of the patients. At least 10 (14%) of the patients developed C. difficile colitis. Additional infection control measures introduced included the use of ward memos, a hand-hygiene awareness campaign, increased environmental cleaning, attention to prescribing practices for antibiotics, increased awareness of diarrheal illness, and early isolation of affected patients. Total use of fluoroquinolones did not change throughout the study period. Despite persistence of this toxin-variant strain, the incidence of C. difficile-associated disease in our institution decreased to fewer than 5 cases per 1,000 admissions. CONCLUSIONS We report on the emergence of a fluoroquinolone- and clindamycin-resistant, toxin A-negative, and toxin B-positive strain of C. difficile associated with an outbreak of C. difficile-associated disease in our institution during a 6-month period. We found that careful attention to improvement of infection control interventions was the most important means of controlling this nosocomial pathogen.

Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples

BackgroundA real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.ResultsThe real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method.ConclusionReal-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.

Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium

Cronobacter species (formerly known as Enterobacter sakazakii) are opportunistic pathogens that can cause necrotizing enterocolitis, bacteraemia and meningitis, predominantly in neonates. Infection in these vulnerable infants has been linked to the consumption of contaminated powdered infant formula (PIF). Considerable research has been undertaken on this organism in the past number of years which has enhanced our understanding of this neonatal pathogen leading to improvements in its control within the PIF production environment. The taxonomy of the organism resulted in the recognition of a new genus, Cronobacter, which consists of seven species. This paper presents an up‐to‐date review of our current knowledge of Cronobacter species. Taxonomy, genome sequencing, current detection protocols and epidemiology are all discussed. In addition, consideration is given to the control of this organism in the manufacturing environment, as a first step towards reducing the occurrence of this pathogen in PIF.

Antimicrobial Resistance in Foodborne Pathogens - A Cause for Concern?

The widespread use of antibiotics in food animal production systems has resulted in the emergence of antibiotic resistant zoonotic bacteria that can be transmitted to humans through the food chain. Infection with antibiotic resistant bacteria negatively impacts on public health, due to an increased incidence of treatment failure and severity of disease. Development of resistant bacteria in food animals can result from chromosomal mutations but is more commonly associated with the horizontal transfer of resistance determinants borne on mobile genetic elements. Food may represent a dynamic environment for the continuing transfer of antibiotic resistance determinants between bacteria. Current food preservation systems that use a combination of environmental stresses to reduce growth of bacteria, may serve to escalate development and dissemination of antibiotic resistance among food related pathogens. The increasing reliance on biocides for pathogen control in food production and processing, heightens the risk of selection of biocide-resistant strains. Of particular concern is the potential for sublethal exposure to biocides to select for bacteria with enhanced multi-drug efflux pump activity capable of providing both resistance to biocides and cross-resistance to multiple antibiotics. Although present evidence suggests that biocide resistance is associated with a physiological cost, the possibility of the development of adaptive mutations conferring increased fitness cannot be ruled-out. Strategies aimed at inhibiting efflux pumps and eliminating plasmids could help to restore therapeutic efficacy to antibiotics and reduce the spread of antibiotic resistant foodborne pathogens through the food chain.

Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

ABSTRACT Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.