Prevalence and characterization of Salmonella spp. among marine animals in the Channel Islands, California

Salmonella enterica is a zoonotic pathogen that has been isolated from free-ranging marine mammals throughout the world, with animals in the Channel Islands of California (USA) showing the highest prevalence. The goal of this study was to determine prevalence, antimicrobial sensitivity and genetic similarity using pulsed-field gel electrophoresis (PFGE) of Salmonella in several non-domestic animal species on San Miguel and San Nicolas Islands. Fecal samples were collected from 90 California sea lion Zalophus californianus pups, 30 northern elephant seal Mirounga angustirostris pups and 87 western gulls Larus occidentalis in the Channel Islands and 59 adult male sea lions in Puget Sound, WA (USA). Salmonella were isolated, identified and serotyped, followed by antimicrobial susceptibility testing and PFGE. Of the California sea lion pups that were sampled on the islands, 21% (n = 19) were positive for Salmonella, whereas no adults males in Puget Sound were positive. Of the northern elephant seal pups sampled, 87% (n = 26) were harboring Salmonella. Only 9% (n = 8) of western gulls were shedding Salmonella, with one of these gulls harboring the only antimicrobial resistant isolate. The serotypes found in these animals were Enteritidis, Montevideo, Newport, Reading, and Saint Paul. The only serotype that showed variation on PFGE was Newport. The pinnipeds of the Channel Islands harbor Salmonella at a higher prevalence than pinnipeds from other geographic areas observed in previous studies. Researchers and veterinarians should exercise increased caution when working with these animals due to the zoonotic potential of Salmonella.     

Aerobic oral and rectal bacteria of free-ranging Steller sea lion pups and juveniles (Eumetopias jubatus) in Alaska

Bacteriologic cultures from oral, rectal, and lesion samples from free-ranging Steller sea lion (SSL, Eumetopias jubatus) pups and juveniles in Alaska (2001-2005) were examined to determine frequency of infection by a specific subset of common and pathogenic aerobic bacteria. Associations between isolated bacteria and age, sex, body condition, location, and sampling season were investigated. Salmonella spp. isolates were further evaluated to determine spatial clustering (n=48) and to identify serovars (n=13) and antimicrobial susceptibility patterns (n=11). We sampled 356 SSL pups (n=272) and juveniles (n=84), and identified 988 isolates of 13 bacterial genera of specific interest. Pasteurella spp. (43.8%), beta-hemolytic Streptococcus spp. (30.6%), and Mannheimia spp. (18.2%) were the most commonly isolated oral bacteria (n=499 isolates), whereas Escherichia coli (47.6%), beta-hemolytic E. coli (32.4%), Salmonella spp. (10.4%), and Campylobacter spp. (7.8%) were the most frequently isolated rectal bacteria (n=460 isolates). Salmonella was most commonly found in pups from western stocks and in samples collected during fall/winter seasons. A significant Salmonella cluster was detected at the Perry Island haulout. Five serovars were isolated: Enteritidis, Infantis, Newport, Reading, and Stanley. Pulsed-field gel electrophoresis provided evidence that Salmonella isolates were most likely being maintained within the SSL population in Alaska.     

A comparison of Salmonella serotypes isolated from New Zealand sea lions and feral pigs on the Auckland Islands by pulsed-field gel electrophoresis

The Salmonella serotypes S. Cerro and S. Newport were isolated from New Zealand sea lions (Phocarctos hookeri) and feral pigs on the Auckland Islands in the New Zealand subantarctic region. The isolates were typed by pulsed-field gel electrophoresis using Xba1 as the restriction enzyme. The isolates were indistinguishable, which suggests that Salmonella infection cycles between sea lions and pigs in this environment. Apart from a previous isolation from a single New Zealand fur seal (Arctocephalus forsteri), S. Newport has not been recorded in any animals from New Zealand, but it is associated with gastroenteritis in humans. Contamination of the marine environment by human waste is a possible source of infection for marine mammals and warrants further investigation.     

Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract

Salmonella enterica subsp. enterica serovar Newport resistant to the extended-spectrum cephalosporins (ESCs) and other antimicrobials causes septicemic salmonellosis in humans and animals and is increasingly isolated from humans, animals, foods, and environmental sources. Mechanisms whereby serovar Newport bacteria become resistant to ESCs and other classes of antimicrobials while inhabiting the intestinal tract are not well understood. The present study shows that 25.3% of serovar Newport strains isolated from the turkey poult intestinal tract after the animals were dosed with Escherichia coli harboring a large conjugative plasmid encoding the CMY-2 beta-lactamase and other drug resistance determinants acquired the plasmid and its associated drug resistance genes. The conjugative plasmid containing the cmy-2 gene was transferred not only from the donor E. coli to Salmonella serovar Newport but also to another E. coli serotype present in the intestinal tract. Laboratory studies showed that the plasmid could be readily transferred between serovar Newport and E. coli intestinal isolates. Administration of a single dose of ceftiofur, used to prevent septicemic colibacillosis, to 1-day-old turkeys did not result in the isolation of ceftiofur-resistant E. coli or Salmonella serovar Newport. There was a remarkable association between serotype, drug resistance, and plasmid profile among the E. coli strains isolated from the poults. This study shows that Salmonella serovar Newport can become resistant to ESCs and other antibiotics by acquiring a conjugative drug resistance plasmid from E. coli in the intestines.     

Survival of Salmonella enterica serovar Newport in manure and manure-amended soils

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log(10), 28 to 64 days for 2 log(10), and 42 to 96 days for 3 log(10) declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.     

Draft Genome Sequences of Eight Salmonella enterica Serotype Newport Strains from Diverse Hosts and Locations

Salmonellosis is a major contributor to the global public health burden. Salmonella enterica serotype Newport has ranked among three Salmonella serotypes most commonly associated with food-borne outbreaks in the United States. It was thought to be polyphyletic and composed of independent lineages. Here we report draft genomes of eight strains of S. Newport from diverse hosts and locations.     

Living in the danger zone: innate immunity to Salmonella

Phagocytic cells, including macrophages, neutrophils and dendritic cells, are critical components of the innate immune response to bacterial pathogens such as Salmonella typhimurium. These cells can have several roles during the early stage of an infection including controlling bacterial replication and producing cytokines and chemokines that activate and recruit additional cells. Macrophages, neutrophils and dendritic cells increase in number early after oral Salmonella infection and produce cytokines important in host survival such as tumor necrosis factor alpha (TNF-alpha). All three phagocytic cell types also harbor bacteria during infection. Natural killer cells, natural killer T cells and T cell receptor alpha beta T cells also respond rapidly to infection and are early sources of interferon-gamma during infection with Salmonella. Studies using infection models with Salmonella are providing a picture of the innate response to bacteria and insight into the role of defined cell types and cytokines important in the transition from innate to adaptive immunity.     

Characterization of antimicrobial resistant Salmonella Kinshasa from dairy calves in Texas

AIM: To determine the prevalence of antimicrobial resistance among Salmonella isolated from a central Texas dairy calf farm that raises animals for dairy-beef production. METHODS AND RESULTS: Salmonella isolates collected from 50 faecal samples were characterized for susceptibility to 20 antimicrobial agents. Seventy per cent of the faecal samples (35 of 50) tested positive for Salmonella, and high rates of resistance to the following drugs that are commonly used for treatment of bacterial enteritis in livestock were observed: ampicillin (88%), apramycin (83%), neomycin (86%), spectinomycin (91%) and oxytetracycline (90%). No resistance to the fluoroquinolone antibiotics was observed. The most prevalent Salmonella serotype was Kinshasha (22 of 35 samples), followed by Agona (4 of 35), Newport (3 of 35), Infantis (2 of 35), Montevideo (2 of 35), Lille (1 of 35) and Newington (1 of 35). The Kinshasa, Agona, Newport and Infantis serotypes all displayed resistance to ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline, and the penta-resistance phenotype was transferable to an Escherichia coli recipient strain. SIGNIFICANCE: Multi-drug resistant Salmonella in dairy calves pose a costly animal health problem and a potential risk to the public health. This study emphasizes the need for alternative, non-antimicrobial intervention strategies for the control of zoonotic pathogens.     

Prevalence and serovars of Salmonella in the feces of free-ranging white-tailed deer (Odocoileus virginianus) in Nebraska

To determine the prevalence and serovars of Salmonella in free-ranging deer, we cultured feces from white-tailed deer (Odocoileus virginianus) harvested by hunters during a regular firearm season in southeastern Nebraska (USA). We recovered Salmonella from 5 (1%; 95% confidence interval: 0.37-2.20%) of 500 samples and identified four different Salmonella enterica serovars [Litchfield (1), Dessau (1), Infantis (2), and Enteritidis (1)]. Although the prevalence of Salmonella in free-ranging deer appears to be low, the serovars recovered are known to be pathogenic to humans and animals.     

Multilocus sequence typing supports the hypothesis that cow- and human-associated Salmonella isolates represent distinct and overlapping populations

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.     

gammadelta T cells may dichotomously modulate infection with avirulent Salmonella choleraesuis via IFN-gamma and IL-13 in mice

To investigate the roles of gammadelta T cells in Salmonella infection, we examined the resolution of an intraperitoneal infection with avirulent Salmonella choleraesuis 31N-1 in mice lacking T-cell-receptor (TCR) alphabeta T cells by disruption of the TCRbeta chain gene (TCRbeta(-/-)). The bacteria in TCRbeta(-/-) mice decreased with kinetics similar to that seen in control mice (TCRbeta(+/+)) after infection. The number of natural killer (NK) cells in the peritoneal cavity increased on day 6 after infection and thereafter decreased in both TCRbeta(-/-) and TCRbeta(+/+) mice, whereas the number of gammadelta T cells, in place of alphabeta T cells, increased remarkably in the peritoneal cavity of TCRbeta(-/-) mice on day 6 after infection. The NK cells from Salmonella-infected TCRbeta(-/-) mice produced interferon-gamma (IFN-gamma) but neither interleukin-4 (IL-4) nor IL-13 in response to immobilized anti-NK1.1 monoclonal antibody (mAb). The gammadelta T cells produced IFN-gamma but neither IL-4 nor IL-13 in response to heat-killed Salmonella, whereas both IFN-gamma and IL-13 but no IL-4 was produced by the gammadelta T cells stimulated with immobilized anti-TCRgammadelta mAb. In vivo administration of anti-NK1.1 mAb inhibited the reduction of Salmonella, whereas anti-TCRgammadelta mAb treatment did not affect the bacterial growth in TCRbeta(-/-) mice after Salmonella infection. However, neutralization of endogenous IL-13 with anti-IL-13 mAb enhanced the bacterial clearance in TCRbeta(-/-) mice after infection. These results suggest that NK1.1(+) cells serve mainly to protect against avirulent Salmonella infection in the absence of alphabeta T cells, whereas gammadelta T cells may play dichotomous roles in Salmonella infection through IFN-gamma and IL-13 in TCRbeta(-/-) mice.     

A role for natural killer cells in intestinal inflammation caused by infection with Salmonella enterica serovar Typhimurium

Acute gastroenteritis caused by Salmonella infection is a significant public health problem. Using a mouse model of this condition, the authors demonstrated previously that the cytokine gamma interferon (IFN-gamma) is required for a normal intestinal inflammatory response to the pathogen. In the present study, these experiments are extended to show that natural killer (NK) cells constitute an early source of intestinal IFN-gamma during Salmonella infection, and that these cells have a significant impact on intestinal inflammation. It was found that infection of mice with Salmonella increased both intestinal IFN-gamma production and the numbers of NK cells in the intestine and mesenteric lymph nodes. NK cells, along with other types of lymphocytes, produced IFN-gamma in response to the bacteria in vitro, while antibody-mediated depletion of NK cells in vivo resulted in a significant reduction in Salmonella-induced intestinal IFN-gamma expression. In a mouse strain lacking NK cells and T and B lymphocytes, intestinal production of IFN-gamma and Salmonella-induced intestinal inflammation were both significantly decreased compared with a strain deficient only in T and B cells. The authors' observations point to an important function for NK cells and NK-derived IFN-gamma in regulating the intestinal inflammatory response to Salmonella.     

Evaluating Gulls as Potential Vehicles of Salmonella enterica Serotype Newport (JJPX01.0061) Contamination of Tomatoes Grown on the Eastern Shore of Virginia

Salmonella enterica serovar Newport pattern JJPX01.0061 has been identified as causing several multistate outbreaks in the last 10 years, primarily due to contamination of tomatoes grown in Virginia. The goal of this study was to evaluate gulls as a potential vehicle of S. Newport pattern 61 contamination for tomatoes grown on the Eastern Shore of Virginia. Gull fecal samples were collected at four sites in eastern Virginia for 3 months (May to July) in 2012, resulting in 360 samples, among which Salmonella was isolated from 62 samples. Twenty-two serotypes and 26 pulsed-field gel electrophoresis DNA fingerprint patterns, including S. Newport pattern 61, were identified. All of the patterns that were isolated multiple times, with the exception of S. Newport patterns JJPX01.0030 and JJPX01.0061, were clustered in time and geographical location. These results strongly suggest that both patterns of S. Newport are endemic to sites on the Eastern Shore where gulls were sampled. This study provides additional information regarding the epidemiology of S. Newport pattern 61 in Virginia and how tomatoes sold interstate may become contaminated in the field.     

Evolution and population structure of Salmonella enterica serovar Newport

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including third-generation cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla(CMY-2) gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.     

Nasal and cloacal bacteria in free-ranging desert tortoises from the western United States

Aerobic bacteria were collected from three free-ranging desert tortoise (Gopherus agassizii) populations in the eastern Mojave Desert (Arizona, Utah; USA) from 1989 to 1993, and from two free-ranging populations in the central Sonoran Desert (Arizona, USA) from 1990 to 1994. Six species of nasal bacteria and 18 species of cloacal bacteria were identified. At least one potential pathogen was found in the nasal cavity (Pasteurella testudinis), and at least two potential pathogens in the cloaca (Pseudomonas spp., Salmonella spp.).     

Detection of Salmonella enterica Subpopulations by Phenotype Microarray Antibiotic Resistance Patterns

Three strains of Salmonella enterica serotype Enteritidis were compared to Salmonella enterica serotype Heidelberg, Salmonella enterica serotype Newport, and Salmonella enterica serovar Typhimurium for growth in the presence of 240 antibiotics arranged within a commercial high-throughput phenotype microarray. The results show that antibiotic resistances were different for subpopulations of serotype Enteritidis separated only by genetic drift.     

Campylobacteriosis, salmonellosis, and shigellosis in free-ranging human-habituated mountain gorillas of Uganda

For conservation purposes and due to growing ecotourism, free-ranging mountain gorillas (Gorilla gorilla beringei) have been habituated to humans. Fecal specimens (n = 62) collected in January 1999 from mountain gorillas of the Bwindi and Mgahinga National Parks, Uganda, were tested for Campylobacter spp., Salmonella spp., and Shigella spp., and the overall prevalence of infection was 19%, 13%, and 6%, respectively. The prevalence of positive specimens was not related to the year of habituation of a gorilla group to humans. Campylobacter spp., Salmonella, and Shigella spp. infections were not distributed equally among the age classes of gorillas; most of the enteropathogens (80%), and all Shigella spp. organisms, S. sonnei, S. boydii, and S. flexneri, were isolated from subadults and adult gorillas with ages ranging from 6.0 to 11.9 yr. The prevalence of Campylobacter spp. and Salmonella spp. infections among human-habituated gorillas has doubled during the last 4 yr, and isolation of Shigella spp. for the first time from mountain gorillas, may indicate enhanced anthropozoonotic transmission of these enteropathogens.     

Survival of Salmonella enterica Serovar Newport in Manure and Manure-Amended Soils

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log₁₀, 28 to 64 days for 2 log₁₀, and 42 to 96 days for 3 log₁₀ declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.     

Isolation of Salmonella spp. from yacare caiman (Caiman yacare) and broad-snouted caiman (Caiman latirostris) from the Argentine Chaco

Presence of Salmonella spp. was evaluated in yacare caiman (Caiman yacare) and broad-snouted caiman (Caiman latirostris) from a ranching facility in the Argentine Chaco. Crocodilian ranching programs are based on captive breeding of wild-harvested eggs and release of excess hatchlings into the wild. Samples for bacterial isolation were collected from 102 captive (35 C. yacare and 67 C. latirostris) and seven free-ranging caiman (four C. yacare and three C. latirositris) between 2001 and 2005 and from three artificially incubated C. yacare wild eggs. Two Salmonella spp. of known zoonotic potential, S. infantis and S. nottingham, were isolated from captive caiman in 2001 and 2002, respectively. This is the first report for S. nottingham in reptiles and of S. infantis in caiman. Salmonella spp. prevalence varied significantly between years, with a 77% prevalence peak in 2002. Although the cause of this increase was not confirmed, we found no correlation with the type of enclosure, caiman species, or body weight. Deteriorated physical condition of caiman hatchlings due to dietary changes in 2002 could have influenced Salmonella spp. shedding. However, external sources such as food, water, or enclosures could not be ruled out. Pathogenic Salmonella spp. present a risk for human infection. Inadvertent introduction of Salmonella spp. or other bacteria into the environment when caiman are released could pose a threat to wild caiman populations. Prophylactic measures to detect and decrease Salmonella spp. presence in caiman ranching facilities are recommended to reduce risk to humans and make caiman-ranching a sound conservation strategy for crocodilian species.     

Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and bla(CMY-2) genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States

Multidrug resistance plasmids carrying the bla(CMY-2) gene have been identified in Salmonella enterica serovars Typhimurium and Newport from the United States. This gene confers decreased susceptibility to ceftriaxone, and is most often found in strains with concomitant resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline. The bla(CMY-2)-carrying plasmids studied here were shown to also carry the florfenicol resistance gene, floR, on a genetic structure previously identified in Escherichia coli plasmids in Europe. These data indicate that the use of different antimicrobial agents, including phenicols, may serve to maintain multidrug resistance plasmids on which extended-spectrum cephalosporin resistance determinants co-exist with other resistance genes in Salmonella.