In-feed use of heavy metal micronutrients in U.S. swine production systems and its role in persistence of multidrug-resistant salmonellae.

The study aimed to characterize the role of heavy metal micronutrients in swine feed in emergence of heavy-metal-tolerant and multidrug-resistant Salmonella organisms. We conducted a longitudinal study in 36 swine barns over a 2-year period. The feed and fecal levels of Cu(2+) and Zn(2+) were measured. Salmonella was isolated at early and late finishing. MICs of copper sulfate and zinc chloride were measured using agar dilution. Antimicrobial susceptibility was tested using the Kirby-Bauer method, and 283 isolates were serotyped. We amplified pcoA and czcD genes that encode Cu(2+) and Zn(2+) tolerance, respectively. Of the 283 isolates, 113 (48%) showed Cu(2+) tolerance at 24 mM and 164 (58%) showed Zn(2+) tolerance at 8 mM. In multivariate analysis, serotype and source of isolates were significantly associated with Cu(2+) tolerance (P < 0.001). Fecal isolates were more likely to be Cu(2+) tolerant than those of feed origin (odds ratio [OR], 27.0; 95% confidence interval [CI], 2.8 to 250; P = 0.0042) or environmental origin (OR, 5.8), implying the significance of gastrointestinal selective pressure. Salmonella enterica serotypes Typhimurium and Heidelberg, highly significant for public health, had higher odds of having >20 mM MICs of Cu(2+) than did "other" serotypes. More than 60% of Salmonella isolates with resistance type (R-type) AmStTeKm (32 of 53) carried pcoA; only 5% with R-type AmClStSuTe carried this gene. czcD gene carriage was significantly associated with a higher Zn(2+) MIC (P < 0.05). The odds of having a high Zn(2+) MIC (≥8 mM) were 14.66 times higher in isolates with R-type AmClStSuTe than in those with R-type AmStTeKm (P < 0.05). The findings demonstrate strong association between heavy metal tolerance and antimicrobial resistance, particularly among Salmonella serotypes important in public health.

Association of multicellular behaviour and drug resistance in Salmonella enterica serovars isolated from animals and humans in Ethiopia.

AIMS: To determine the association between multicellular behaviour, integron status and antibiotic resistance among 87 Ethiopian Salmonella enterica isolates of animal and human origin. METHODS AND RESULTS: Isolates were characterized for their biofilm forming ability, antimicrobial susceptibility and the presence and characteristics of a class 1 integron and Salmonella genomic island 1 (SGI1). The majority of isolates grown at environmental temperatures (20°C) exhibited robust biofilm formation (72·4%) and displayed RDAR colony morphology on Congo red agar plates. The presence of a class 1 integron correlated with the extent of drug resistance and ability to exhibit multicellular behaviour. CONCLUSIONS: Although cellulose production and RDAR morphology correlated with increased multicellular behaviour, neither was required for biofilm formation. Contrary to previous reports, colony morphology was generally consistent within a serovar. No integrons were detected in isolates deficient for multicellular behaviour, indicating a potential role of bacterial community formation in transfer of genetic elements among environmental isolates. SIGNIFICANCE AND IMPACT OF STUDY: Infection by Salm. enterica is a major public health problem worldwide. The dominance of multidrug resistance and multicellular behaviour in Salmonella isolates of Ethiopian origin highlights a need for integrated surveillance and further detailed phenotypic and molecular studies of isolates from this region.

Prevalence of porcine noroviruses, molecular characterization of emerging porcine sapoviruses from finisher swine in the United States, and unified classification scheme for sapoviruses.

Noroviruses (NoVs) and sapoviruses (SaVs) are important human pathogens. Although the involvement of porcine NoVs in disease in pigs is unclear, they are genetically and antigenically closely related to human NoVs. Human NoV-like strains have been detected in pigs, raising public health concerns of potential interspecies transmission. Porcine SaVs are highly diverse and emerging in swine populations. Recently, at least three new genogroups of porcine SaVs have been proposed. In this study, we tested 413 pooled fecal samples collected from apparently healthy finisher pigs in North Carolina swine farms during 2009. Reverse transcription (RT)-PCR coupled hybridization assays were performed to detect known porcine NoVs. The overall prevalence of porcine NoVs determined was 18.9% based on this method. Samples were then tested by RT-PCR targeting the 5' end of the capsid region for genogroup II (GII) NoVs, a group which includes human NoVs, followed by sequence analysis. All NoVs identified belonged to typical porcine NoV genotypes, and no human NoV-like strains were detected in specimens from these pigs. Porcine NoV-negative samples (n = 335) were subsequently screened using universal calicivirus primers, and 17 SaV strains were confirmed by sequencing. Based on the partial RNA-dependent RNA polymerase (RdRp) region, they clustered with GIII, GVII, and GVIII and with currently unclassified SaVs. According to analysis of the complete capsid sequences, 7 representative strains clustered with GVII, GVIII, and GIX? SaVs. We tentatively classified SaVs into 14 genogroups based on the complete capsid protein VP1. In summary, porcine NoVs and highly divergent SaVs were present in North Carolina finisher pigs.

Epidemiology and genotypic characteristics of methicillin-resistant Staphylococcus aureus strains of porcine origin.

The main goal of this study was to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), particularly livestock-associated MRSA (LA-MRSA) in pigs and pork. The genotypic relatedness of isolates on the farm, at slaughter, and at the retail level was assessed. Paired nasal and perianal swab samples were collected from 10 cohorts of market-age pigs (24 pigs per cohort) and carcasses at slaughterhouse, and pork samples were collected at retail. Staphylococci were isolated using selective enrichment method. Isolates were tested for antimicrobial resistance by broth microdilution. Duplex PCR was used to confirm MRSA using species-specific (nuc) and methicillin resistance (mecA) genes. The clonal relatedness of isolates was determined using pulsed-field gel electrophoresis (PFGE), Staphylococcus protein A (spa) typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec element (SCCmec) typing. MRSA was detected in 5 of the 10 cohorts (50%), with the prevalence ranging from 0% to 12.5% per cohort. Of all the pigs sampled on the farm before they went to market, 3% (7/240) were MRSA positive. A higher prevalence of MRSA was detected at holding pens at the slaughterhouse (11% [27/240]). MRSA was also detected in 2% (4/235) of the carcasses and 4% (5/135) of the retail pork. While the isolates appear predominantly to be highly clonal, PFGE had a relatively higher discriminatory power (discriminatory index [DI] = 0.624). Four genotypic clusters were identified by PFGE; of the four clusters, clonal type B was predominant across the farm-to-retail continuum. MLST findings revealed that sequence type 5 (ST5) was the most predominant subtype (32/50). The livestock-associated MRSA (clonal complex 398 [CC398] or sequence type 398 [ST398]) was the second common type (12/50) and was detected at all stages from farm to retail. Nine of the 50 (18%) MRSA isolates belonged to spa type 539/t034 that were of ST398 based on MLST. The results of this study confirm that MRSA, including LA-MRSA, is common in herds of swine in Ohio and hereby shown to persist in the farm to processing and retail continuum.

Characterization and prevalence of a new porcine Calicivirus in Swine, United States.

Real-time reverse transcription PCR revealed that new St-Valerien-ike porcine caliciviruses are prevalent (2.6%-80%; 23.8% overall) in finisher pigs in North Carolina. One strain, NC-WGP93C, shares 89.3%-89.7% genomic nucleotide identity with Canadian strains. Whether these viruses cause disease in pigs or humans or are of food safety concern requires further investigation.

Salmonella enterica in commercial swine feed and subsequent isolation of phenotypically and genotypically related strains from fecal samples.

The purpose of this study was to determine the occurrence and genotypic relatedness of Salmonella enterica isolates recovered from feed and fecal samples in commercial swine production units. Of 275 feed samples, Salmonella was detected in 10 feed samples that originated from 8 of 36 (22.2%) barns, with a prevalence of 3.6% (10/275 samples). In fecal samples, a prevalence of 17.2% was found at the early finishing stage (1,180/6,880 samples), with a significant reduction in prevalence (7.4%) when pigs reached market age (392/5,321 samples). Of the 280 Salmonella isolates systematically selected for further characterization, 50% of the feed isolates and 55.3% of the isolates of fecal origin showed similar phenotypes based on antimicrobial resistance patterns and serogrouping. About 44% of the isolates were multidrug resistant. Pulsed-field gel electrophoresis (PFGE) genotyping grouped the 46 representative isolates into five genotypic clusters, of which four of the clusters consisted of genotypically related isolates recovered from feed and fecal samples. The occurrence of genotypically related and, in some cases, clonal strains, including multidrug-resistant isolates in commercially processed feed and fecal samples, suggests the high significance of commercial feed as a potential vehicle of Salmonella transmission.

Tracking salmonella contamination in various watersheds and phenotypic and genotypic diversity.

Salmonella enterica is an important foodborne pathogen, and contamination of surface and ground water that may result from various human activities, such as animal production and urbanization, may contribute to the public health burden. The aims of this study was to determine the sources of Salmonella contamination in four different types of watersheds and to assess the relative contribution of multidrug-resistant strains. Eighty-six water samples collected from four different watershed systems, including those impacted by swine production (n = 12), residential/industrial (n = 34), crop agriculture (n = 12), and forestry (n = 28), were cultured for Salmonella and further characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis genotyping. Salmonella prevalence was high in all four watersheds: residential/industrial area (58.8%), forestry (57.1%), crop agriculture (50%), and swine production (41.7%). Majority of the Salmonella isolates (87.1%) were pansusceptible. Multidrug resistance up to eight antimicrobials (R-type: AmStTeAxChCeKmGm) was detected in water samples that originated from swine production systems only. Serovars identified included Anatum, Gaminara, and Inverness (18.3% each) and Muenchen and Newport (8.7% each), Bredeny (7.6%), and Montevideo (6.8%). Pulsed-field gel electrophoresis analysis indicated genotypic relatedness among Salmonella recovered from residential/industrial and forestry-associated watersheds (genotypic cluster types A, C, D, E, F, G, H, and J), sites with relatively close geographic proximity. Swine-production-associated isolates were distinctly different from the others (genotypic cluster types B and I), corroborating the phenotypic findings. Overall, the findings suggest that all the various watersheds, including natural forest, remain important contributors of Salmonella contamination. While swine-production-associated water samples were not found to have a disproportionately high prevalence, it was the most important reservoir of multidrug-resistant strains.

Class 1 integrons and resistance gene cassettes among multidrug resistant Salmonella serovars isolated from slaughter animals and foods of animal origin in Ethiopia.

The present study was undertaken to identify and characterize integrons and integrated resistance gene cassettes among multidrug resistant (MDR) Salmonella isolates from slaughter animals and food products of animal origin in Ethiopia. A total of 98 epidemiologically unrelated Salmonella isolates comprising 13 serovars were characterized using serotyping, phage typing, antimicrobial resistance testing and the pulsed-field gel electrophoresis (PFGE) method. Integron-PCR was used to detect the presence of class 1 and class 2 integrons in the MDR strains. The associated individual resistance gene cassettes were identified using specific PCRs and DNA sequencing. The location of the integrons was determined by Southern blot hybridization analysis. Among the Salmonella serovars, a high level of antimicrobial resistance was found to streptomycin (82.6%), tetracycline (75.5%), sulfamethoxazole (60.2%), spectinomycin (53.1%), ampicillin (42.8%), nalidixic acid (34.7%), nitrofurantoin (30.6%), trimethoprim (27.5%), gentamicin (20.4%) and ciprofloxacin (19.4%). Class 1 integrons were detected in 53.1% of the MDR isolates comprising serovars Anatum, Braenderup, Kentucky, Saintpaul and Typhimurium. Of the class 1 integron positive isolates 61.5% harboured the integron-associated gene cassettes: aadA2, aadA2+bla(PSE-1), dfrA1-aadA1 and dfrA12-orf-aadA2 (amplicon sizes 1000 bp, 1000+1200 bp, 1600 bp and 1900 bp, respectively). The chromosomally located aadA2 and aadA2+bla(PSE-1) resistance gene cassettes occurred exclusively in S. Typhimurium DT104 isolates, the other cassettes were found on large plasmids in several serovars. An aacCA5-aadA7 gene cassette array (amplicon size 1600 bp) was exclusively found in all MDR S. Kentucky strains of R type Str/SpeSmxGenNalAmpTetCipCef and this integron was shown to be chromosomally located. Results of the present study indicate that class 1 integrons carrying gene cassettes, which confer resistance to different classes of antimicrobials such as aminoglycosides, beta-lactams and trimethoprim are widespread among the MDR Salmonella serovars isolated from slaughter animals and food products of animal origin in Ethiopia indicating the important role of these genetic elements in the dissemination of multidrug resistance.

Distribution, quantitative load and characterization of Salmonella associated with swine farms in upper-northern Thailand.

This study was conducted to analyze the prevalence and quantitative loads of Salmonella spp. on pig farms in Chiang Mai, Lamphun, Thailand to assess loading levels before slaughtering. The serotype diversity, antimicrobial-resistance pattern and pulse-field type of Salmonella spp. were also characterized to assess the dynamic propagation of the pathogen. The Salmonella-positive prevalence was 246/805 (30.56%), and the quantitative loads varied from 1.48 ˜ 4.04 Log10MPN/g, with a mean ± standard deviation of 2.11 ± 0.57. AMP/S/TE (ampicillin/streptomycin/tetracycline) was the highest frequency antimicrobial resistance pattern found in this study. In addition, Salmonella Rissen was the primary serotype in this region. PFGE results indicated the occurrence of infection by cross contamination among pig farms. Our study showed that pork is easily contaminated with this pathogen. Farm control programs must be based on strict biosecurity and hygienic measures, which could further reduce the contamination pressure at slaughterhouses or retail shops.

Methicillin-resistant Staphylococcus aureus in pigs and farm workers on conventional and antibiotic-free swine farms in the USA.

Much uncertainty remains about the origin and public health implications of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA). This study aimed to investigate the occurrence and prevalence of MRSA in general and LA-MRSA in particular in pigs and farm workers in five states. We collected nasal swabs from pigs and farm workers at 45 swine herds (21 antibiotic-free herds; 24 conventional herds) in Illinois, Iowa, Minnesota, North Carolina and Ohio. MRSA was isolated from 50 of 1085 pigs (4.6%) and 31 of 148 (20.9%) of farm workers. MRSA-positive pigs and people were clustered in four conventional swine farms in Iowa and Illinois. Based on genotyping, spa type t034, a common livestock associated variant, was predominant among both human and swine isolates. These results confirm the presence of LA-MRSA in pigs and swine farm workers in the USA, but the prevalence found is relatively low compared with European studies.

Comparison of California Mastitis Test (CMT), Somatic Cell Counts (SCC) and bacteriological examinations for detection of camel (Camelus dromedarius) mastitis in Ethiopia.

A total of 956 quarter milk samples from 253 traditionally managed lactating camels were collected aseptically from Negele (Borena Region), Dire Dawa, and Gewane (Afar Region), Ethiopia, according to multi-stage sampling. The quarter milk samples were subjected to California Mastitis Test (CMT), Somatic Cell Counts (SCC) and bacteriological examinations. Five hundred and seventy one (59.7%) quarter milk samples had microorganisms. Of these, 428 (75.0%) had isolates that were identified as major pathogens (MAP) and 143 (25.0%) as minor pathogens (MIP). A positive correlation was found between CMT scores and bacteriological classes (MAP, MIP) (p-value = 0.00). Strong correlation (p-value = 0.00) between CMT scores and SCC was recorded. The differences among the median log SCC of bacteriological classes (MAP, MIP) were not significant (p-value = 0.24). Similarly, the application of the cut-off level of 2.5 x 10(5) ml(-1) indicated less agreement (p-value = 0.32) for bacteriological classes MAP and MIP.

Occurrence of Salmonellae in retail raw chicken products in Ethiopia.

A cross-sectional study was undertaken to determine the presence and prevalence of salmonellae in retail raw chicken meat and giblets (gizzard and liver) in supermarkets in Addis Ababa (Ethiopia). A total of 301 samples (244 chicken meat, 32 gizzards and 25 livers) were collected from 22 randomly selected supermarkets and examined for the presence of Salmonella. For the isolation and identification of salmonellae, the technique recommended by the International Organization for Standardization (ISO 6579, 1998) was used. Salmonellae were detected from 54 (17.9%) of the 301 samples examined. Chicken meat and giblet samples in 68.2% (15/22) of the supermarkets were contaminated with salmonellae. The contamination level of Salmonella was higher in chicken giblets as compared to chicken meat, which were respectively 12.3%, 53.1% and 28.0% in chicken meat, gizzard and liver samples. Out of the 54 Salmonella isolates, nine different serotypes were identified: Salmonella Braenderup (31.5%), S. Anatum (25.9%), S. Saintpaul (14.8%), S. Uganda (11.1%), S. Haifa, S. Group B, S. Rough form and S. Typhimurium (each 3.7%) and S. Virchow (1.8%). The high level of Salmonella contamination of chicken meat and giblets observed in the present study indicated the need in an improvement in the microbiological quality of retail chicken in Ethiopia.

Antimicrobial resistance pattern of Salmonella serotypes isolated from apparently healthy slaughtered camels (Camelus dromedarius) in eastern Ethiopia.

A total of 714 samples consisting of faeces, mesenteric lymph nodes, liver, spleen, abdominal and diaphragmatic muscles (each 119) were collected from November 2001 to April 2002 from apparently healthy slaughtered camels (Camelus dromedarius) in eastern Ethiopia. One hundred sixteen (16.2%) Salmonella strains belonging to 16 different serovars were isolated. All Salmonella strains isolated were examined for antimicrobial resistance to 17 selected antimicrobials. The minimum inhibitory concentration (MIC) values were determined by the microdilution broth test. Fifty-two (44.8%) of the Salmonella isolates were resistant to one or more antimicrobials. Thirty-nine of the 52 (75%) resistant Salmonella serovars exhibited multiple resistance to up to eight different antimicrobials. Among the serovars tested, S. Typhimurium, S. Heidelberg, S. Braenderup and S. Hadar displayed multiple resistance mainly to streptomycin (35.3%), spectinomycin (28.4%), sulfamethoxazole (25.0%), ampicillin (24.1%), trimethoprim (22.4%), trimethoprim/sulfamethoxazole (18.9%), tetracycline (12.9%) and colistin (11.2%). All Salmonella strains tested were susceptible to ciprofloxacin, nalidixic acid, gentamicin, kanamycin and neomycin. The present study showed the importance of camels as a potential source of single and multiple resistant Salmonella strains to different antimicrobials that are also used in the public health sector for the treatment of different bacterial diseases in Ethiopia.