Molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Japan

Aims:  To investigate the prevalence of integrons and antimicrobial resistance genes in Salmonella recovered from animals in Japan.
Methods and Results:  Forty-eight out of ninety-four (51·1%) Salmonella isolates showed multidrug resistance phenotypes and harboured at least one antimicrobial resistance gene. Twenty-two out of forty-seven (46·8%) Salmonella enterica serovar Typhimurium that were multidrug-resistant were of definitive phage type DT104. Class 1 integrons were identified in 34/94 isolates (36·2%): 21 isolates containing two gene cassettes, aadA2 and bla _PSE–1, and 13 containing one gene cassette, aadA1 , aadA2 or bla _PSE–1. Class 2 integrons containing estX - sat2 - aadA1 gene cassettes were only identified in Salmonella Enteritidis. The β-lactamase-encoding gene, bla _TEM, was only detected in S. Typhimurium. The plasmid-mediated quinolone resistance gene, qnrS1 , was identified in S. Typhimurium and Salmonella Thompson.
Conclusions:  Our results characterized integrons and antimicrobial resistance genes in Salmonella of animal origin. To the best of our knowledge, this is the first report of qnrS in Salmonella from Japan and also the first report of qnrS in S . Thompson.
Significance and Impact of the Study:  Little is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals. This study provides useful data on the incidence of integrons and resistance genes in Salmonella of animal origin.     

Distribution and genotypic characterization of Salmonella serovars isolated from tropical seafood of Cochin, India

Aims:  To determine the distribution of Salmonella serovars in seafood and to examine the intraserovar genetic variations in Salmonella enterica subsp. enterica serovar Rissen and Salmonella Weltevreden by polymerase chain reaction (PCR)-ribotyping and enterobacterial repetitive intergenic consensus (ERIC)-PCR methods.
Method and Results:  A total of 417 seafood samples collected over 2003–2006 from fishing harbours and fish markets of Cochin (India) was studied for presence of Salmonella serovars. Seafood samples were analysed for the presence of Salmonella by Bacteriological Analytical Manual (BAM), U.S. Food & Drug Administration (USFDA) method. The study indicated that 23·2% of the seafood samples were positive for Salmonella and a total of 241 Salmonella isolates comprising of 27 different serovars were isolated from seafood. S. Weltevreden, Salmonella Rissen, Salmonella Typhimurium and Salmonella Derby were found to be the most predominant serovars in seafood. PCR-ribotypes and ERIC-PCR profiles showed multiple genotypic profiles for S. Rissen and S. Weltevreden in seafood and the level of discrimination indices obtained was at 0·974 for S. Rissen and 0·988 for S. Weltevreden, respectively.
Conclusion:  The study highlighted the major Salmonella serovars in the seafood of Cochin (India) and molecular fingerprinting pattern revealed genetic variation among S. Rissen and S. Weltevreden.
Significance and Impact of the Study:  Widespread occurrence of Salmonella contamination in seafood and multiple clones of S. Rissen and S. Weltevreden detected in seafood, thus, indicated the diverse routes of Salmonella contamination in seafood.     

Sanitation of faeces from source-separating dry toilets using urea

Aim:  To develop a reliable and simple method to produce safe fertilizers from human excreta using urea for sanitation of faeces.
Methods and Results:  Urea was added to faecal matter (17% dry matter) at concentrations of 0·5–2% (w/w) and inactivation of Salmonella enterica subspecies 1 serovar Typhimurium (Salm. Typhimurium), Enterococcus spp . and the Salm. Typhimurium bacteriophage 28B was monitored at 14, 24 and 34°C. Urea additions enhanced inactivation and inactivation rates were positively related to increasing NH₃ (aq) concentration and temperature. Salm. Typhimurium was the most sensitive of the organisms studied, while Enterococcus spp. showed more persistence, especially at lower temperatures. The bacteriophage was the most resistant organism studied.
Conclusions:  Salmonella reduction levels that meet requirements for safe reuse of faeces as fertilizer (i.e. 6 log₁₀ reduction) can be achieved for 1% urea within 2 months at 14°C or within 1 week at 24°C and 34°C.
Significance and Impact of the Study:  The relationships between organism inactivation rates and temperature, ammonia and pH were identified. Urea treatment proved to be a robust and efficient option for safe recycling of plant nutrients.     

Microarray analysis of antimicrobial resistance genes in Salmonella enterica from preharvest poultry environment

Aims:  To detect antimicrobial resistance genes in Salmonella isolates from turkey flocks using the microarray technology.
Methods and Results:  A 775 gene probe oligonucleotide microarray was used to detect antimicrobial resistance genes in 34 isolates. All tetracycline-resistant Salmonella harboured tet(A) , tet(C) or tet(R) , with the exception of one Salmonella serotype Heidelberg isolate. The sul1 gene was detected in 11 of 16 sulfisoxazole-resistant isolates. The aadA , aadA1 , aadA2 , strA or strB genes were found in aminoglycoside-resistant isolates of Salm. Heidelberg, Salmonella serotype Senftenberg and untypeable Salmonella . The prevalence of mobile genetic elements, such as class I integron and transposon genes, in drug-resistant Salmonella isolates suggested that these elements may contribute to the dissemination of antimicrobial resistance genes in the preharvest poultry environment. Hierarchical clustering analysis demonstrated a close relationship between drug-resistant phenotypes and the corresponding antimicrobial resistance gene profiles.
Conclusions:  Salmonella serotypes isolated from the poultry environment carry multiple genes that can render them resistant to several antimicrobials used in poultry and humans.
Significance and Impact of the Study:  Multiple antimicrobial resistance genes in environmental Salmonella isolates could be identified efficiently by microarray analysis. Hierarchical clustering analysis of the data was also found to be a useful tool for analysing emerging patterns of drug resistance.     

Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes

A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates.     

Antimicrobial resistance and virulence determinants in European Salmonella genomic island 1-positive Salmonella enterica isolates from different origins

Salmonella genomic island 1 (SGI1) contains a multidrug resistance region conferring the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype encoded by bla(PSE-1), floR, aadA2, sul1, and tet(G). Its increasing spread via interbacterial transfer and the emergence of new variants are important public health concerns. We investigated the molecular properties of SGI1-carrying Salmonella enterica serovars selected from a European strain collection. A total of 38 strains belonging to S. enterica serovar Agona, S. enterica serovar Albany, S. enterica serovar Derby, S. enterica serovar Kentucky, S. enterica serovar Newport, S. enterica serovar Paratyphi B dT+, and S. enterica serovar Typhimurium, isolated between 2002 and 2006 in eight European countries from humans, animals, and food, were subjected to antimicrobial susceptibility testing, molecular typing methods (XbaI pulsed-field gel electrophoresis [PFGE], plasmid analysis, and multilocus variable-number tandem-repeat analysis [MLVA]), as well as detection of resistance and virulence determinants (PCR/sequencing and DNA microarray analysis). Typing experiments revealed wide heterogeneity inside the strain collection and even within serovars. PFGE analysis distinguished a total of 26 different patterns. In contrast, the characterization of the phenotypic and genotypic antimicrobial resistance revealed serovar-specific features. Apart from the classical SGI1 organization found in 61% of the strains, seven different variants were identified with antimicrobial resistance properties associated with SGI1-A (S. Derby), SGI1-C (S. Derby), SGI1-F (S. Albany), SGI1-L (S. Newport), SGI1-K (S. Kentucky), SGI1-M (S. Typhimurium), and, eventually, a novel variant similar to SGI1-C with additional gentamicin resistance encoded by aadB. Only minor serovar-specific differences among virulence patterns were detected. In conclusion, the SGI1 carriers exhibited pathogenetic backgrounds comparable to the ones published for susceptible isolates. However, because of their multidrug resistance, they may be more relevant in clinical settings.     

Case report of clinical salmonellosis by Salmonella Typhimurium that occurred in Portuguese children

Aims: Aim of this study is to characterize clinical isolates of Salmonella Typhimurium that occurred in Portuguese children on the basis of their virulence and antimicrobial resistance profiles and pulsed‐field gel electrophoresis typing and to analyse possible strain relatedness. Methods and Results: Different Salmonella serotypes were isolated from clinical cases of salmonellosis that had occurred in two Portuguese hospitals (a total of 259 isolates). All Salm. Typhimurium strains, with the age of the patients known, (total of 26 isolates) were selected for this study. These isolates were characterized for their virulence gene profiles (agfA, iroB, slyA, hin/H2, spv), antimicrobial resistance profiles and investigated for the occurrence of multidrug‐resistant Salm. Typhimurium DT 104 by PCR. Salmonella isolates showed high rates of resistance to four or more antibiotics, 100% resistance to sulfadiazine and a high percentage of strains with the resistance profile of Salm. Typhimurium DT 104, two of them with this phage type (determined by PCR). A relationship between some clusters and their resistance and virulence profiles was detected, each cluster having the same profile. Conclusions: This study showed high‐antibiotic resistance of the Salmonella strains investigated, and the presence of multidrug‐resistant Salm. Typhimurium DT104 in infections of Portuguese children. Significance and Impact of the Study: Study is based on regarding the increase in antibiotic resistance by Salmonella strains isolated from infections in Portuguese children and on the presence of Salm. Typhimurium DT 104 circulating in Portugal.     

Characterization of antimicrobial susceptibility and virulence genes of Salmonella serovars collected at a commercial turkey processing plant

Aims:  To determine the antimicrobial susceptibility profiles, distribution of class 1 integrons, virulence genes and genes encoding resistance to tetracycline ( tetA , tetC , tetD and tetE ) and streptomycin ( strA , strB and aadA1 ) in Salmonella recovered from turkeys.
Methods and Results:  The antimicrobial susceptibility of 80 isolates was determined using National Antimicrobial Resistance Monitoring System. The distribution of resistance genes, class 1 integrons and virulence genes was determined using PCR. Resistances to tetracycline (76·3%) and streptomycin (40%) were common. Sixty-two (77·5%) isolates displayed resistance against one or more antimicrobials and 33 were multi-drug resistant. tetA was detected in 72·5% of the isolates, while tetC , tetD and tetE were not detected. The strA and strB genes were detected in 73·8% of the isolates. Two isolates possessed class 1 integrons of 1 kb in size, containing the aadA1 gene conferring resistance to streptomycin and spectinomycin. Fourteen of the virulence genes were detected in over 80% of the isolates.
Conclusions:  This study shows that continuous use of tetracycline and streptomycin in poultry production selects for resistant strains. The Salmonella isolates recovered possess significant ability to cause human illness.
Significance and Impact of the Study:  Information from this study can be employed in guiding future strategies for the use of antimicrobials in poultry production.     

Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The β-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.     

Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan

Aims:  To compare bacterial populations and antimicrobial resistance patterns between clinical and sewage isolates from a regional hospital in northern Taiwan. The dissemination of antibiotic-resistant bacteria from hospital compartments to the hospital sewage treatment plant was examined.
Methods and Results:  A total of 1020 clinical isolates and 435 sewage isolates were collected between July and September 2005. The percentages of Gram-negative bacteria from the clinical and sewage isolates were 87·2% and 91·0%, respectively ( P  =   0·033). Escherichia coli were the leading bacterial isolates in both groups. Antimicrobial susceptibility testing showed a significant difference ( P  <   0·001) in resistance to ampicillin (85·6% vs 94·1%), ampicillin/sulbactam (31·7% vs 55·4%), cefazolin (29·2% vs 71·5%) and cefuroxime (20·7% vs 61·9%) between clinical and sewage coliform isolates, respectively.
Conclusions:  The sewage isolates had higher antimicrobial resistance rates than the clinical isolates from the same hospital.
Significance and Impact of the Study:  The low efficacy of the hospital sewage treatment may contribute to the dissemination of multidrug resistant bacteria from this hospital compartments to the environment. Practices which limit the disposal of antimicrobial agents into the wastewater system may be the possible measure to prevent the selection of multidrug-resistant bacteria from sewage treatment plants.     

Pulsed-field gel electrophoretic identification of Salmonella enterica serovar Typhimurium live vaccine strain Zoosaloral H

Salmonella enterica subsp. enterica serovar Typhimurium ( Salm. Typhimurium) live vaccine strain Zoosaloral H was characterized by pulsed-field gel electrophoresis (PFGE). Each of the two suitable restriction enzymes, Xba I and Spe I, produced a unique restriction fragment pattern for this live vaccine strain which was not shared by field isolates of the same serovar. The characteristic fragment pattern proved to be stable during a 22 month observation period and was also not altered after animal passage of the vaccine strains. Thus PFGE analysis proved to be a helpful tool in the identification of Salm. Typhimurium live vaccine strain Zoosaloral H and its differentiation from wild-type isolates of the same serovar.     

Selection of Salmonella typhimurium as an indicator for pathogen regrowth potential in composted biosolids

In order to select a suitable indicator for monitoring the pathogen regrowth potential of composted biosolids, the growth kinetics of selected bacteria were investigated. Growth parameters of six serovars of Salmonella and three strains of Escherichia coli in sterilized compost were compared. Seeded Salmonella and E. coli grew rapidly, reaching population densities of more than 10(8) g-1 after 30 h of incubation. The specific growth rates of Salmonella serovars and E. coli strains were similar and varied from 0.49 to 0.55 h-1. The specific growth rate of the Salm. Typhimurium isolates was significantly higher than the other bacterial strains. It was concluded that an antibiotic-resistant strain of Salm. Typhimurium can be used as an indicator for a pathogen regrowth potential test.     

Prevalence and serotypes of Salmonella associated with goats at two Australian abattoirs

Aims:  This study was carried out to determine the prevalence and serotype of Salmonella in goats presented for slaughter.
Methods and Results:  A total of 121 goats were examined for the presence of Salmonella in matching rumen, faecal and carcass samples. Samples were analysed for the presence of Salmonella following the Australian Standard AS 1766.2.5-1991. Salmonella was isolated from 56 (46·3%) faecal samples, 55 (45·5%) rumen samples and 35 (28·9%) carcass samples. The dominant serotypes isolated were Salmonella serotype Saintpaul (31%), Salmonella serotype Typhimurium (13%) and Salmonella serotype Chester (11%).
Conclusions:  Salmonella was isolated from at least one of the three sample sites in 68% of animals. Carcase contamination with faeces, compared with rumen liquor, is a greater hazard for Salmonella contamination of goat carcases. Goat meat is a potential source of Salmonella serovars associated with human disease.
Significance and Impact of the Study:  Goat carcases contaminated with Salmonella during slaughter could be a source of food-borne disease if consumed raw or inadequately cooked, or may be a source of cross-contamination to other foods.     

Comparative evaluation of methods for counting surviving biofilm cells adhering to a polyvinyl chloride surface exposed to chlorine or drying

Aim:  To compare techniques for assessing biofilm populations previously subjected, or not, to inimical treatment by chlorine or drying.
Methods and Results:  Four sonication treatments and swabbing were compared on Salmonella  Typhimurium or Pseudomonas fluorescens biofilms grown on polyvinyl chloride (PVC). The apparatuses emitting the highest ultrasound energy were the most efficient except on Salm. Typhimurium biofilms subjected to drying: a lethal effect, leading to an underestimation of at least 1·5 log (CFU cm^–2) was observed with the more aggressive treatment. The differences between the highest count and the swabbing counts ranged from 0·3 log (CFU cm^–2) (untreated Salm. Typhimurium) to 1·7 log (CFU cm^–2) (chlorine treated Ps. fluorescens ). Impedance measurements, used to assess populations without detaching any cells showed that the calibration curves that were built up from data obtained with suspended cells plus PVC slides were not appropriate.
Conclusions:  High energy ultrasound techniques designed either for in vitro or in situ studies proved efficient in assessing the number of attached CFU. However, the right treatment duration has to be carefully established before using high energy ultrasound techniques.
Significance and impact of the study:  The effectiveness of a cleaning and disinfection regime inferred from data obtained after swabbing may be greatly underestimated.     

Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal

AIMS: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal. METHODS AND RESULTS: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent. CONCLUSIONS: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.     

Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant

Aims:  To isolate environmental bacteria capable of transforming fluoroquinolones to inactive molecules.
Methods and Results:  Bacteria were isolated from the aerobic liquor of a wastewater treatment plant on a medium containing norfloxacin (100 mg l⁻¹). Twenty-two isolates were highly resistant (minimal inhibitory concentration: 6·25−200 μg ml⁻¹) to five fluoroquinolones and six of them were positive by PCR amplification for the aminoglycoside resistance gene aac(6')-Ib. Of these, only Escherichia coli strain LR09 had the ciprofloxacin-acetylating variant gene aac(6')-Ib-cr ; HPLC and mass spectrometry showed that this strain transformed both ciprofloxacin and norfloxacin by N -acetylation. This bacterium also had mutations in the quinolone-resistance determining regions of the gyrA and parC genes.
Conclusions:  An E. coli isolate from wastewater, which possessed at least two distinct fluoroquinolone resistance mechanisms, inactivated ciprofloxacin and norfloxacin by N -acetylation.
Significance and Impact of the Study:  This is the first report of N -acetylation of fluoroquinolones by an aac(6')-Ib-cr -containing bacterium from an environmental source.     

Detection of a Salmonella enterica serovar California strain spreading in spanish feed mills and genetic characterization with DNA microarrays

We performed an epidemiological study on Salmonella isolated from raw plant-based feed in Spanish mills. Overall, 32 different Salmonella serovars were detected. Despite its rare occurrence in humans and animals, Salmonella enterica serovar California was found to be the predominant serovar in Spanish feed mills. Different typing techniques showed that isolates of this serovar were genetically closely related, and comparative genomic hybridization using microarray technology revealed 23 S. enterica serovar Typhimurium LT2 gene clusters that are absent from serovar California.     

Effects of yeast probiotic formulation on viability, revival and protection against infection with Salmonella enterica ssp. enterica serovar Typhimurium in mice

Aims:  To compare the effects of five yeast probiotic formulations on viability, revival and washout kinetic in the digestive tract of mice, and the protection against an experimental infection with Salmonella enterica serovar Typhimurium.
Methods and Results:  The number of viable cells in five commercial probiotic products codified as A, B, C and D ( Saccharomyces boulardii – lyophilized) and E ( Saccharomyces cerevisiae – aqueous suspension) was determined, as well as revival and washout kinetic in mouse intestine. Protective capacity was evaluated by survival rate and histopathology of liver and intestine of mice treated with each product and then challenged with Salm . Typhimurium.
Conclusions:  Product A contained the highest number of viable cells and, fed to mice, gave the highest counts of viable yeasts and the longest persistence in faeces. Probably as a consequence, the highest survival and protection of intestinal and hepatic tissues were observed when product A was used for mouse treatment. Product E showed low counts in the formulation and was not recovered from mouse intestine.
Significance and Impact of the Study:  Formulation (lyophilization or aqueous suspension) is an important factor for revival and survival of a probiotic product in vivo and consequently for its protective properties.     

Effect of selected antimicrobial compounds on the radiosensitization of Salmonella Typhi in ground beef

Aims:  In this study, we extended our previous work to determine the efficiency of antimicrobial compounds in increase of relative radiosensitivity of Salmonella Typhi in medium fat ground beef (23% fat) by testing 41 different essential oils (EOs), oleoresins and food sauces.
Methods and Results:  Ground beef samples inoculated with Salmonella Typhi (10⁶ CFU g⁻¹ ) were treated with each antimicrobial compound at a concentration of 0·5% (w/w). Then, the samples (25 g each) were packaged under air and irradiated in a ⁶⁰Co irradiator at doses from 0 to 1·75 kGy. Radiosensitivity was evaluated by calculating relative radiation sensitivity, defined as the ratio of radiation D ₁₀ value in the absence/presence of antimicrobial compound.
Conclusions:  Depending on the compound tested, the addition of antimicrobial compound decreased the D ₁₀ value of Salmonella Typhi, resulting in an increase of the radiation sensitivity up to more than four times. Among these antimicrobial compounds, Chinese cinnamon EO, clove EO and trans -cinnamaldehyde were most effective to increase the radiosensitivity of Salmonella Typhi in ground beef.
Significance and Impact of the Study:  These observations demonstrate that some active compounds can function as radiosensitizers of Salmonella Typhi.