Serotype Agona Research Articles

Effect of low doses of biocides on the susceptibility of Listeria monocytogenes and Salmonella enterica to various antibiotics of clinical importance

The use of subinhibitory concentrations of biocides in food processing environments requires special attention because it is related to potential increases in antibiotic resistance. In this study, we determined the effect of exposure to low doses of four biocides (sodium hypochlorite, SHY; benzalkonium chloride, BZK; peracetic acid, PAA; trisodium phosphate, TSP) on the resistance to 10 antibiotics and on the hydrophobicity of the cellular surface of a strain of Listeria monocytogenes serotype 1/2 b (LM) and a strain of Salmonella enterica serotype Agona (SA), both of meat origin. The cultures were exposed at 37 °C in tryptone soya broth to increasing concentrations of the biocides, starting with half the minimum inhibitory concentration (MIC/2) and incrementing by 1.5 times the concentration until growth was no longer observed, calculating the MIC of the antibiotics before (control cultures) and after exposure. After exposure to TSP, LM was able to grow in the presence of a concentration of the biocide 2.53 times higher than the MIC of unexposed cultures. No adaptation was observed for SHY, BZK or PAA. SE demonstrated adaptation to BZK (it tolerated a concentration 1.13 times higher than the MIC for the unadapted strain) and PAA (2.53 times). LM cultures presented increased resistance (from susceptibility to reduced susceptibility, from susceptibility to resistance, or from reduced susceptibility to resistance) to erythromycin (strains exposed to BZK, PAA and TSP), chloramphenicol (all compounds), tetracycline (BZK) and fosfomycin (all compounds). Regarding SA, after exposure its resistance to gentamicin (all compounds) and enrofloxacin (BZK, PAA and TSP) increased. The cell surface hydrophobicity (determined through the microbial adhesion to solvents -MATS- test) increased (LM exposed to PAA and TSP; SA exposed to BZK) or decreased (SA exposed to PAA) after contact with the biocides. These findings suggest that the use of biocides at subinhibitory concentrations can contribute to the increase in bacterial resistance to antibiotics, in addition to modifying the hydrophobicity of the cellular surface, which is related to the capacity of bacteria to form biofilm.

Is There a Shift in Salmonella Diversity Among Poultry in Northern India?

The present study was conducted to determine the serotype diversity of Salmonella among poultry in northern India. A total of 101 poultry droppings from 30 farms in the Jammu and Kashmir union territory were analyzed. Nineteen isolates of Salmonella were obtained, and these belonged to four serotypes: Salmonella enterica enterica serotype Kentucky (n = 3), Salmonella enterica enterica serotype Infantis (n = 5), Salmonella enterica enterica serotype Agona (n = 4), and Salmonella enterica enterica serotype Typhimurium (n= 7). The study has isolated some Salmonella serotypes that are infrequently reported in India. Some of the isolated serotypes are reported to be endemic for human nontyphoidal salmonellosis cases in the region. Whether this indicates a shift in the serotype pattern in poultry in the region needs to be investigated further. Nevertheless, the study clearly indicates the risk of foodborne salmonellosis associated with consumption of contaminated poultry and poultry products in the region.

Comparative Analysis Using Pulsed-Field Gel Electrophoresis Highlights a Potential Transmission of Salmonella Between Asymptomatic Buffaloes and Pigs in a Single Farm.

Buffaloes and pigs play an important epidemiological roll in the Salmonella infection cycle, and asymptomatic animals can act as key component in the dissemination of the disease by horizontal, vertical, and cross-species transmission. Our study aimed and was able to confirm evidences of a cross-species transmission of Salmonella Agona between asymptomatic buffaloes and pigs. Also, we described Salmonella infection within the pig production phases, involving serotypes Agona, Senftenberg and Schwarzengrund. Rectal samples were collected from Jafarabadi buffaloes (n = 25) and Piau pigs (n = 32), located on a single farm. Salmonella Agona was isolated from lactating buffaloes, gilts, pregnant sows, and weaned pigs, Salmonella Schwarzengrund from lactating sows and Salmonella Senftenberg from gilts, pregnant sows, lactating sows, and weaned pigs. Pulsed-field Gel Electrophoresis protocol (PFGE) was performed and revealed four different profiles. Profile 1 (Salmonella Agona), isolated from a pregnant sow, a gilt and two lactating buffaloes, revealed a indistinguishable PFGE pattern, confirming evidences of potential cross-species transmission. Profile 2 (Salmonella Agona), 3 (Salmonella Senftenberg), and 4 (Salmonella Schwarzengrund), isolated from pigs, revealed important indistinguishable PFGE patterns, evidencing Salmonella infection within the pig production phases. Considering the epidemiological relevance of buffaloes and pigs in the cycle of Salmonella infection, confirmation of a potential cross-species transmission of Salmonella Agona and potential Salmonella infection within the pig production phases highlights the importance of the correct establishment of preventive health strategies in farms, in special the importance of avoiding contact between buffaloes and pigs, since cross-species transmission can occur, increasing the risk of spreading the disease.

Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 μm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 μm3 ± 2131.0 μm3 (S. enterica), 58,418.3 μm3 ± 5944.9 μm3 (L. monocytogenes), 68,020.8 μm3 ± 5812.3 μm3 (MRSA) and 59,280.0 μm3 ± 4032.9 μm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.

Salmonella spp. in Wild Free-Living Birds from Atlantic Forest Fragments in Southern Bahia, Brazil.

Wild animals have an ecological function and can serve as sentinels to identify infectious agents and as indicators of environmental health. Among the zoonotic pathogens, Salmonella spp. deserve special attention due to their high worldwide prevalence and their ubiquity of hosts. With the aim of investigating the presence of Salmonella spp. in wild birds from the Atlantic Forest in southern Bahia, Brazil, we collected 114 fecal samples of wild birds (14 families) between 2016 and 2017. Fecal samples were collected by means of cloacal swab and subjected to microbiological culture to isolate and serotype Salmonella spp. specifically. Antibiotic susceptibility was determined using the disk diffusion test protocol. Only one bird, Ceratopipra rubrocapilla, tested positive for Salmonella enterica subsp. enterica serotype Agona, which is the first record for this bird species. This isolate exhibited intermediate sensitivity to amikacin and gentamicin and sensitivity to the other 13 antibiotics tested. Results may indicate environmental preservation since the studied areas had minimal human activity and good sanitary quality. Despite the low prevalence, it is necessary to monitor wildlife and establish disease control and surveillance systems, especially for zoonotic diseases.

Simultaneous growth, survival and death: The trimodal behavior of Salmonella cells under osmotic stress giving rise to “Phoenix phenomenon”

Time-lapse microscopy methods were used to monitor growth, survival and death of Salmonella enterica serotype Agona individual cells on solid laboratory medium (tryptone soy agar) in the presence of various salt concentrations (0.5%, 3.5%, 4.5% and 5.7% NaCl). The results showed a highly heterogeneous behavior. As NaCl concentration increased, the distribution of the first division time was shifted to higher values and became wider. The mean first division time increased from 1.8 h at 0.5% NaCl to 5.48 h, 16.2 h, and 35.9 h at 3.5%, 4.5% and 5.7% NaCl, respectively. The concentration of NaCl in the growth medium also affected the ability of the cells to divide. The percentage of cells able to grow decreased from 88.9% at 0.5% NaCl to 66.5%, 32.8%, and 6.9% at 3.5%, 4.5% and 5.7% NaCl, respectively. In the latter case (5.7% NaCl), 74 cells out of 406 cells tested (18%) died with mean time to death 5.03 h and standard deviation 6.70 h. To investigate the effect of the behavior of individual cells on the dynamics of the whole population, simulation analysis was used. The simulation results showed that the simultaneous growth, survival and death of cells observed under osmotic stress can lead to a total population behavior known as the “Phoenix” phenomenon. The simulation findings were confirmed by validation experiments using both viable counts and time lapse microscopy. The results of the present study show the high heterogeneity of individual cell responses and the complexity in the behavior of microbial populations at conditions approaching the boundaries of growth.

Visualization and quantification of the cellular and extracellular components of Salmonella Agona biofilms at different stages of development.

Salmonella is a major food-borne pathogen able to persist in food processing environments because of its ability to form biofilms. A Salmonella enterica serotype Agona isolate from poultry (S24) was grown at 37°C in biofilms for up to 144 hours (H144) in attachment to polystyrene surfaces. Biofilm structures were examined at different stages in their development (H3, H24, H48, H72, H96 and H144) using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9), dead cells (propidium iodide), proteins (fluorescein isothiocyanate isomer I), lipids (DiD’oil), α-polysaccharides (concanavalin A, tetramethylrhodamine conjugate), and β-polysaccharides (calcofluor white M2R). Strain S24 developed a robust biofilm at H72 (biovolume of 166,852.5 ± 13,681.8 μm3 in the observation field of 16,078.2 μm2). The largest biovolume of live cells was also detected at H72 (128,110.3 ± 4,969.1 μm3), decreasing thereafter, which was probably owing to the detachment of cells prior to a new phase of colonization. The percentage of dead cells with regard to total cells in the biofilms increased throughout the incubation, ranging from 2.3 ± 1.1% (H24) to 44.2 ± 11.0% (H144). Proteins showed the greatest biovolume among the extracellular components within the biofilms, with values ranging from 1,295.1 ± 1,294.9 μm3 (H3) to 19,186.2 ± 8,536.0 μm3 (H96). Maximum biovolume values of 15,171.9 ± 660.7 μm3 (H48), 7,055.3 ± 4,415.2 μm3 (H144), and 2,548.6 ± 1,597.5 μm3 (H72) were observed for β-polysaccharides, α-polysaccharides and lipids, respectively. A strong (P < 0.01) positive correlation was found between the total biovolume of biofilm and the biovolume of live cells, proteins and β-polysaccharides, which may serve as useful markers of biofilm formation. The present work provides new insights into the formation of S. Agona biofilms. Our findings may contribute to the designing of reliable strategies for preventing and removing these bacterial communities.

A Multistate Outbreak of Human Salmonella Agona Infections Associated With Consumption of Fresh, Whole Papayas Imported From Mexico-United States, 2011.

Nontyphoidal Salmonella causes ~1 million food-borne infections annually in the United States. We began investigating a multistate outbreak of Salmonella serotype Agona infections in April 2011. A case was defined as infection with the outbreak strain of Salmonella Agona occurring between 1 January and 25 August 2011. We developed hypotheses through iterative interviews. Product distribution analyses and traceback investigations were conducted. The Food and Drug Administration (FDA) tested papayas from Mexico for Salmonella. We identified 106 case patients from 25 states. Their median age was 21 years (range, 1-91). Thirty-nine of 61 case patients (64%) reported Hispanic/Latino ethnicity; 11 of 65 (17%) travelled to Mexico before illness. Thirty-two of 56 case patients (57%) reported papaya consumption. Distribution analyses revealed that three firms, including Distributor A, distributed papaya to geographic areas that aligned with both the location and timing of illnesses. Traceback of papayas purchased by ill persons in four states identified Distributor A as the common supplier. FDA testing isolated the outbreak strain from a papaya sample collected at distributor A and from another sample collected at the US-Mexico border, destined for distributor A. FDA isolated Salmonella species from 62 of 388 papaya import samples (16%). The investigation led to a recall of fresh, whole papayas from Distributor A and an FDA import alert for all papayas from Mexico. This is the first reported Salmonella outbreak in the United States linked to fresh, whole papayas. The outbreak highlights important issues regarding the safety of imported produce.

Multiplex PCR–Based Serogrouping and Serotyping of Salmonella enterica from Tonsil and Jejunum with Jejunal Lymph Nodes of Slaughtered Swine in Metro Manila, Philippines

Food poisoning outbreaks and livestock mortalities caused by Salmonella enterica are widespread in the Philippines, with hogs being the most commonly recognized carriers of the pathogen. To prevent and control the occurrence of S. enterica infection in the country, methods were used in this study to isolate and rapidly detect, differentiate, and characterize S. enterica in tonsils and jejuna with jejunal lymph nodes of swine slaughtered in four locally registered meat establishments (LRMEs) and four meat establishments accredited by the National Meat Inspection Services in Metro Manila. A total of 480 samples were collected from 240 animals (120 pigs from each type of meat establishment). A significantly higher proportion of pigs were positive for S. enterica in LRMEs (60 of 120) compared with meat establishments accredited by the National Meat Inspection Services (38 of 120). More S. enterica-positive samples were found in tonsils compared with jejuna with jejunal lymph nodes in LRMEs, but this difference was not significant. A PCR assay targeting the invA gene had sensitivity that was statistically similar to that of the culture method, detecting 93 of 98 culture-confirmed samples. Multiplex PCR-based O-serogrouping and H/Sdf I typing revealed four S. enterica serogroups (B, C1, D, and E) and six serotypes (Agona, Choleraesuis, Enteritidis, Heidelberg, Typhimurium, and Weltevreden), respectively, which was confirmed by DNA sequencing of the PCR products. This study was the first to report detection of S. enterica serotype Agona in the country.

Antimicrobial Susceptibility and Molecular Typing ofSalmonellaAgona Isolated from Humans and Other Sources

Salmonella enterica serotype Agona (Salmonella Agona) has been among the top 10 serotypes that cause human diarrheal diseases in China. A total of 95 Salmonella Agona (67 from humans, and 28 from animals, food of animal origins, and environmental sources) recovered in Shanghai, China from 2005 to 2011 were subjected to antimicrobial susceptibility testing and molecular subtyping using pulsed-field gel electrophoresis (PFGE). Approximately 68.4% of the Salmonella Agona isolates were pansusceptible to 15 antimicrobial agents tested, and 4 isolates (4.21%) were resistant to at least 3 antimicrobials. PFGE analysis resulted in 41 unique patterns, of which 4 major PFGE patterns (X3, X4, X5, and X6) were grouped together at 96.1% similarity. Isolates of the four patterns included those from food (pork, beef, and chicken) and humans. Our findings showed that the same clones of Salmonella Agona were recovered from human patients and food, and that food of animal origin was potentially a major vehicle of Salmonella Agona in human salmonellosis in Shanghai.

Individual cell heterogeneity as variability source in population dynamics of microbial inactivation

A statistical modeling approach was applied for describing and evaluating the individual cell heterogeneity as variability source in microbial inactivation. The inactivation data (Nt vs time) of Salmonella enterica serotype Agona, with initial concentration N0 = 109 CFU/ml in acidified tryptone soy broth (pH 3.5), were transformed to (N0 − Nt)/N0 vs time leading to the cumulative probability distribution of the individual cell inactivation times (ti), which was further fitted to a variety of continuous distributions using @Risk software. The best-fitted ti distribution (Gamma) was used to predict the inactivation of S. Agona populations of various N0 using Monte Carlo simulation, with the number of iterations in each simulation being equal to N0 and the number of simulations representing the variability of the population inactivation behavior. The Monte Carlo simulation results for a population with N0 = 10,000 CFU/ml showed that the variability in the predicted inactivation behavior is negligible for concentrations down to 100 cells. As the concentration decreases below 100 cells, however, the variability increases significantly. The results also indicated that the D-value used in deterministic first order kinetic models is valid only for large populations. For small populations, D-value shows a high variability, originating from individual cell heterogeneity, and, thus, can be better characterized by a probability distribution rather than a uniform value. Validation experiments with small populations confirmed the variability predicted by the statistical model. The use of the proposed approach to quantify the variability in the inactivation of mixed microbial populations, consisting of subpopulations with different probability distributions of ti, was also demonstrated.

Response of restraint stress-selected lines of Japanese quail to heat stress and Escherichia coli challenge

Japanese quail selected for divergent corticosterone response to restraint stress were evaluated for their susceptibility to heat stress and challenge with Escherichia coli. These quail lines are designated as high stress (HS), low stress (LS), and the random-bred control (CS) lines. Heat stress (35°C, 8 h/d) began at 24 d until the end of the study at 39 d. Birds were challenged with an aerosol spray containing 2 × 10(9) cfu of E. coli at 25 and 32 d. At 38 d, the surviving birds were necropsied and the intestinal tract was screened for both Salmonella and Campylobacter. Body weights of the CS birds were higher than both HS and LS at 17, 25, and 32 d. At 32 d, there was no difference in mortality between males and females and the CS line had higher mortality compared with the LS line with the HS line being intermediate. At 38 d, females of the CS line that were both heat stressed and challenged had a mortality incidence of 25%, which was significantly higher than male birds of the same line and treatment (5.3%). There was an increased incidence in Salmonella enterica serotype Agona isolation after heat stress, with the LS birds having less isolation than the HS birds. Mean corticosterone levels of male birds were not significantly affected by line, heat stress, or E. coli challenge; however, the LS line subjected to heat stress had one-third the level of the HS line, a difference identical to that seen in the original selection for response to restraint stress.

Draft Genome Sequences of Three Salmonella enterica Serotype Agona Strains from China

Salmonellosis has been one of the major contributors to the global public health burden. Salmonella enterica serotype Agona has ranked among the top 10 and top 20 most frequent Salmonella serotypes isolated from human sources in China and the United States, respectively. We report draft genomes of three S. Agona strains from China.

A Recurrent, Multistate Outbreak of Salmonella Serotype Agona Infections Associated with Dry, Unsweetened Cereal Consumption, United States, 2008

An outbreak of Salmonella enterica serotype Agona infections associated with nationwide distribution of cereal from Company X was identified in April 2008. This outbreak was detected using PulseNet, the national molecular subtyping network for foodborne disease surveillance, which coincided with Company X's voluntary recall of unsweetened puffed rice and wheat cereals after routine product sampling yielded Salmonella Agona. A case patient was defined as being infected with the outbreak strain of Salmonella Agona, with illness onset from 1 January through 1 July 2008. Case patients were interviewed using a standard questionnaire, and the proportion of ill persons who reported eating Company X puffed rice cereal was compared with Company X's market share data using binomial testing. The Minnesota Department of Agriculture inspected the cereal production facility and collected both product and environmental swab samples. Routine surveillance identified 33 case patients in 17 states. Of 32 patients interviewed, 24 (83%) reported eating Company X puffed rice cereal. Company X puffed rice cereal represented 0.063% of the total ready-to-eat dry cereal market share in the United States at the time of the investigation. Binomial testing suggested that the proportion of exposed case patients would not likely occur by chance (P < 0.0001). Of 17 cereal samples collected from case patient homes for laboratory testing, 2 (12%) yielded Salmonella Agona indistinguishable from the outbreak strain. Twelve environmental swabs and nine product samples from the cereal plant yielded the outbreak strain of Salmonella Agona. Company X cereal was implicated in a similar outbreak of Salmonella Agona infection in 1998 with the same outbreak strain linked to the same production facility. We hypothesize that a recent construction project at this facility created an open wall near the cereal production area allowing reintroduction of Salmonella Agona into the product, highlighting the resilience of Salmonella in dry food production environments.

Molecular characterization of antimicrobial resistance in non-typhoidal salmonellae associated with systemic manifestations from India

Extended-spectrum cephalosporins and fluoroquinolones are essential antimicrobials for treating invasive salmonellosis, although emerging resistance to these antimicrobials is of growing concern, especially in India. Therefore, a study was conducted to characterize the antimicrobial susceptibility phenotypes, types of extended-spectrum β-lactamase (ESBL) gene plasmids and serological relationships of 21 non-typhoidal Salmonella isolates from patients who attended three different hospitals in India from 2006 to 2008. The isolates were cultured from stool, blood and cerebrospinal fluid samples obtained from patients presenting with diarrhoea and accompanying systemic manifestations such as fever, vomiting and meningism. Non-typhoidal Salmonella isolates were investigated using serotyping and antimicrobial susceptibility testing. PCR screening was also performed to detect the β-lactamase, qnr and aac(6')-Ib-cr genes and class 1 integrons. Sequencing for quinolone resistance mutations and plasmid replicon typing were also performed. An antimicrobial resistance microarray was used for preliminary screening and identification of bla(TEM) and bla(SHV) genes, and phenotypic testing for the presence of efflux pumps was also performed. Ten out of 21 isolates (48%) possessed the extended-spectrum cephalosporin resistance phenotype, with PCR amplification and sequencing revealing that isolates possessed TEM-1, SHV-12, DHA-1, OXA-1-like and CTX-M-15 ESBL genes. FII(s) plasmid replicons were detected in seven isolates (33%). The involvement of efflux pumps was detected in four isolates (19%) resistant to ciprofloxacin. It was concluded that SHV-12-carrying Salmonella serotype Agona may play an important role in ESBL-mediated resistance in non-typhoidal salmonellae in India. The very high percentage (48%) of ESBL-producing non-typhoidal salmonellae isolated from these patients represents a real and immediate challenge to the effective antimicrobial therapy of Salmonella infections associated with systemic manifestations. Continued surveillance for the presence of ESBL-producing (non-typhoidal) salmonellae in India is essential.

Phenotype MicroArray™ in the metabolic characterisation of Salmonella serotypes Agona, Enteritidis, Give, Hvittingfoss, Infantis, Newport and Typhimurium

The Phenotype MicroArray (PM) technology was used to study the metabolic characteristics of 29 Salmonella strains belonging to seven serotypes of S. enterica spp. enterica. Strains of serotypes Typhimurium (six strains among definite phage types DTs 1, 40 and 104) and Agona (two strains) were tested for 949 substrates, Enteritidis (six strains of phage type PT1), Give, Hvittingfoss, Infantis and Newport strains (two of each) were tested for 190 substrates and seven other Agona strains for 95 substrates. The strains represented 18 genotypes in pulsed-field gel electrophoresis (PFGE). Among 949 substrates, 18 were identified that could be used to differentiate between the strains of those seven serotypes or within a single serotype. Unique metabolic differences between the Finnish endemic Typhimurium DT1 and Agona strains were detected, for example, in the metabolism of D-tagatose, D-galactonic acid gamma-lactone and L-proline as a carbon source. Thus, the PM technique is a useful tool for identifying potential differential markers on a metabolic basis that could be used for epidemiological surveillance.

Effects of a commercially available vaccine against Salmonella enterica serotype Newport on milk production, somatic cell count, and shedding of Salmonella organisms in female dairy cattle with no clinical signs of salmonellosis

To determine effects of vaccination with siderophore receptor and porin (SRP) proteins derived from Salmonella enterica serotype Newport on milk production, somatic cell count, and shedding of Salmonella organisms in female dairy cattle. 180 female Holsteins. Cattle were randomly assigned to receive Salmonella Newport SRP vaccine or control solution. Vaccine or control solution was injected 45 to 60 days before parturition, and cattle received a second dose 14 to 21 days before parturition. Milk production was monitored for the first 90 days of lactation. Feces for isolation of Salmonella and blood samples for detection of antibodies against Salmonella Newport were collected at day of first injection and at days 7 to 14 and 28 to 35 of lactation. Cattle inoculated with Salmonella Newport vaccine produced significantly more milk (1.14 kg/d), compared with cattle injected with the control solution. Cattle administered the vaccine had significantly higher concentrations of circulating antibody against Salmonella Newport SRP proteins at 7 to 14 days and 28 to 35 days of lactation. Salmonella Newport was not recovered; however, Salmonella enterica serotype Agona was recovered from 31 (20.3%) cattle, but likelihood of recovery did not differ significantly between vaccinates and control cattle. Administration of a vaccine against Salmonella Newport SRP proteins to healthy dairy cattle prior to parturition increased milk production, even in cattle without detectable shedding of Salmonella Newport or clinical signs of salmonellosis. Additional research is needed to clarify the mechanisms by which productivity was improved.

Two Consecutive Large Outbreaks of Salmonella enterica Serotype Agona Infections in Infants Linked to the Consumption of Powdered Infant Formula

An increase of isolation of Salmonella Agona was observed in January through February 2005 among infants in France. Case-control study, food trace-back and microbiologic investigations were promptly carried out. A total of 141 confirmed cases <12 months of age were identified. Most had diarrhea (99%; bloody 56%) and fever (75%) and 36% were hospitalized for 5 days on average and none died. In the case-control analysis, all 23 cases and none of the 23 controls had consumed powdered formula of brand A (P < 10(-5)). Active follow up of all cases showed that after the withdrawal of formula A, cases that had consumed formula A decreased rapidly, but new cases had consumed another formula (brand B). The trace-back found that 5 batches of formula B had been manufactured on the same production line as formula A. Forty-four cases were linked to formula A and 92 to formula B. All routine controls performed by the producers were negative for Salmonella. However, enhanced microbiologic investigations yielded S. Agona in one of 176 samples of formula A, in 4 of 27 tins of formula B consumed by cases and in 6 of 420 environmental swabs from the production line. All clinical, food and environmental isolates were of the same pulsed-field gel electrophoresis profile. Powdered infant formulas are not sterile products and may contain low levels of Salmonella. Routine microbiologic controls are insufficient to detect a low-grade contamination, which may cause serious illness and outbreaks among infants.

Effects of Different Molting Procedures on Incidence of Salmonella Infection in Flocks of Naturally Contaminated Laying Hens in a Commercial Egg-Producing Farm by Detection of Yolk Antibodies to Salmonella in Eggs

Indirect enzyme-linked immunosorbent assays (ELISAs) have been applied to detect immunoglobulin Y antibodies to different serotypes of Salmonella in the yolks of chicken eggs with heat-extracted antigens of Salmonella enterica serotypes Agona (SA), Cerro (SC), Enteritidis (SE), Montevideo (SM), and Putten (SP). The egg yolk samples examined were classified as positive if their ELISA absorbance values exceeded the value for eggs from specific-pathogen-free flocks by more than two standard deviations. Of 30 egg yolk samples from three flocks vaccinated with a killed SE vaccine, 29 were antibody positive by the ELISA assay for the SE antigen. Four to 29 of the 29 yolk samples showed positive results for the other serovars, although the absorbance values for SE were higher than those obtained for the other serotypes in each of the yolk samples. All 30 yolks from three flocks that were not administered any SE vaccines were found to be antibody negative for SE, and two samples were determined to be positive for SC. Thirty-nine or 40 eggs were obtained from each of four layer flocks in a commercial egg production farm where the laying houses were naturally contaminated with SA, SC, SM, SP, Salmonella serovar Infantis (SI), and untypeable strains. The ELISA absorbance values for SM in the egg yolks obtained from the two flocks molted through feed withdrawal when the birds restarted laying were significantly (P < 0.05) higher than those observed in the yolks obtained before the molt. In egg yolks from the two other flocks that were molted through a wheat bran diet, there was no significant difference between the absorbance values before and after the molt. The observations in the present study provide further evidence to suggest that a molt initiated through the administration of a wheat bran diet can reduce the risk for Salmonella problems in a commercial egg-producing setting.

Salmonella and Shigella in Freshly Squeezed Orange Juice, Fresh Oranges, and Wiping Cloths Collected from Public Markets and Street Booths in Guadalajara, Mexico: Incidence and Comparison of Analytical Routes

A survey of the presence of Salmonella and Shigella in freshly squeezed orange juice and related samples was conducted in Guadalajara, Mexico. One hundred samples of freshly squeezed orange juice were collected from 49 street booths and 51 small food service establishments. In addition, 75 fresh orange samples, each consisting of five orange units, and 75 wiping cloths were collected from the same establishments from which juice had been collected. Salmonella was isolated from 14, 20, and 23% of samples of orange juice, orange surfaces, and wiping cloths collected from street vendors, while Shigella was isolated from 6, 17, and 5% of these samples. In general, the frequency of isolation of these pathogens in samples from juice serving establishments at public markets was significantly lower than that found among street vendors (P < 0.05). Salmonella enterica serotypes Agona, Typhimurium, and Anatum were found in orange juice, fresh oranges, and wiping cloth samples, while serotype Mexico was found on fresh oranges and in wiping cloths and serotypes Muenchen and Panama were found only in wiping cloth samples. Regarding Shigella species, Shigella sonnei was found in all three types of sample tested; Shigella dysenteriae was found in juice and orange samples, Shigella boydii in orange and wiping cloth samples, and Shigella flexneri on oranges only. Thirty-one percent and 39% of the juice samples showed aerobic plate counts of ≥5.0 log CFU/ml and Escherichia coli counts of >3.0 log CFU/ml, respectively. These high counts may indicate poor sanitation and potential exposure to fecal contamination either in the raw materials or during the orange-crushing and juice-serving process. These data may be useful for a further risk assessment of Salmonella or Shigella in unpasteurized, freshly squeezed juice.