Antimicrobial and disinfectant resistance of Salmonella Heidelberg from Brazilian flocks did not increase for ten years (2006-2016) / A resistência a desinfetantes e a antimicrobianos não aumentou em um período de 10 anos (2006 a 2016) em Salmonella Heidelberg isoladas de granjas avícolas brasileiras

Salmonella is a major cause of foodborne illness worldwide, and poultry and its derived products are the most common food products associated with salmonellosis outbreaks. Some countries, including Brazil, have experienced an increased prevalence of Salmonella Heidelberg among their poultry flocks. Some isolates have also presented high resistance to antimicrobial agents and persist in the poultry farm environment. This study aimed to compare the susceptibility of S. Heidelberg strains isolated in 2006 with those isolated in 2016 against disinfectants and antimicrobial agents. The results showed that all the strains were highly susceptible to sodium hypochlorite, regardless of the conditions and year of isolation. Resistance to benzalkonium chloride varied according to the conditions applied, but not to the year of isolation. Increased antimicrobial resistance from 2006-2016 was observed only for tetracycline. The results suggest that the antimicrobial and disinfectant resistance of S. Heidelberg did not increase for ten years (2006-2016). However, further analysis should include a larger number of S. Heidelberg isolates from poultry origin and additional antimicrobial agents for more precise conclusions about the increasing in antimicrobial resistance in the last years.(AU)

Salmonella é uma das principais causas das doenças transmitidas por alimento em todo o mundo, e a carne de frango e produtos derivados são os principais alimentos associados com surtos de salmonelose em humanos. Alguns países, incluindo o Brasil, têm observado um aumento da ocorrência de Salmonella Heidelberg nas suas granjas avícolas. Além disto, alguns isolados têm apresentado alta resistência aos antimicrobianos e têm persistido no ambiente de produção avícola. Neste contexto, o objetivo deste estudo foi comparar a susceptibilidade de cepas de S. Heidelberg isoladas em 2006 com aquelas isoladas em 2016 contra desinfetantes e agentes antimicrobianos. Os resultados demonstraram que as cepas foram altamente resistentes a hipoclorito de sódio, independentemente das condições e do ano de isolamento. A resistência ao cloreto de benzalcônio variou de acordo com as condições testadas, mas não com o ano de isolamento. Um aumento da resistência aos antimicrobianos de 2006 a 2016 foi observado apenas para tetraciclina. Os resultados sugerem que a resistência aos desinfetantes e aos antimicrobianos não aumentou em um período de dez anos (2006-2016). Entretanto, novas análises devem incluir um número maior de cepas de S. Heidelberg isoladas de fontes avícolas e outros agentes antimicrobianos para uma conclusão mais precisa sobre o aumento da resistência antimicrobiana nos últimos anos.(AU)

Detección de serovares de Salmonella en terneros de crianza artificial de la región lechera Mar y Sierras, Argentina / Detection of serovars of Salmonella in artificially reared calves in Mar y Sierras Dairy Basin, Argentina

La salmonelosis es una de las enfermedades bacterianas que afectan el tracto digestivo de los terneros y provocan en ellos diarrea. Con el objetivo de estudiar la prevalencia de los distintos serovares de Salmonella en terneros de crianza artificial y determinar la asociación con signos diarreicos, se realizó un estudio epidemiológico con diseno transversal en la región lechera Mar y Sierras, ubicada en la Provincia de Buenos Aires (Argentina). Mediante hisopado de mucosa rectal, se muestrearon 726 terneros en período de crianza distribuidos en 50 establecimientos lecheros de dicha zona, se incluyeron animales con signos diarreicos y sin estos. Los aislamientos identificados como Salmonella spp. fueron tipificados utilizando antisueros poli- y monovalentes dirigidos contra antígenos somáticos, flagelares y capsulares (Vi). Salmonella spp. se detectó en el 36% de los establecimientos y los serovares hallados fueron S. Mbandaka, S. Anatum, S. Typhimurium, S. Dublin, S. Montevideo, S. Meleagridis, S. Newport, S. Seftemberg, S. subesp.16,7:z1, S. Infantis y S. Give. El 5,5% de los terneros fueron positivos y aquellos terneros con signología diarreica presentaron 5,9 veces más probabilidad de estar infectados con Salmonella spp. que aquellos que no tuvieron signos. La edad de los terneros positivos osciló desde un día hasta 53 días de vida; la mayor frecuencia se detectó al segundo día de nacidos. Se concluye que 11 serovares de Salmonella están presentes en más de un tercio de los establecimientos lecheros de la región lechera Mar y Sierras y que estos serovares mostraron estar asociados a la existencia de signos diarreicos en los terneros, sobre todo a la presencia de moco en las heces. La prevalencia de Salmonella fue mayor en terneros de menos de 21 días de vida.

Salmonellosis in calves is a bacterial disease that affects their digestive tract causing diarrhea. A cross-sectional epidemiological study was carried out with the aim of studying the prevalence of various serovars of Salmonella in calves and their relationship with diarrhea signs. The study was conducted in Mar and Sierras Dairy Basin located in Buenos Aires province, Argentina. Seven hundred and twenty six calves both with diarrhea signs or not were sampled by rectal mucosa swab in 50 dairy farms during the rearing period. Isolates identified as Salmonella spp. were classified using polyvalent and monovalent antisera against somatic, flagellar and capsule antigens (Vi). Salmonella spp. was found in 36% of the farms and serotypes were: S. Mbandaka, S. Anatum, S. Typhimurium, S. Dublin, S. Montevideo, S. Meleagridis, S. Newport, S. Seftemberg, S. subesp. 16,7:z1, S. Infantis, S. Give. A percentage of 5.5% calves was positive and calves showing diarrheal signs were 5.9 times more likely to be infected with Salmonella spp. than those having no signs. The age of positive calves ranged from the first day of life to 53; the second day being the most frequent time. In conclusion, 11 Salmonella serovars were detected in one out of 3 dairy farms in Mar and Sierras Dairy Basin, and not only were these serovars associated with diarrhea signs including the presence of mucus in feces, but they were also more prevalent among calves aged up to 21 days.

Manejo sanitário de aviário de pequeno porte de frangos de corte e presença de Salmonella spp / Sanitary management of small broiler house and the presence of Salmonella spp

A Salmonella spp. um dos principais patógenos na avicultura e têm emergido como um crescente problema econômico e de saúde pública. Objetivou-se avaliar o manejo sanitário de um aviário de pequeno porte e sua relação com a presença de Salmonella spp. Em dois lotes, do ambiente, foram coletadas amostras para análises microbiológicas na entrada dos pintinhos (D0), no 28º (D28) e no 60º (D60) após recepção, feita a aferição das temperaturas, e realizado formulário de acompanhamento das boas práticas de produção. A análise dos dados foi descritiva. Verificou ausência de Salmonella spp. em todas as amostras e valores adequados de temperatura. Portanto, o eficiente monitoramento dos programas de Boas Práticas de Fabricação (BPF) e Procedimentos Padrão de Higiene Operacional (PPHO) pode assegurar o produto final.

A common Salmonella Enteritidis sequence type from poultry and human gastroenteritis in Ibagué, Colombia / Un tipo de secuencia común de Salmonella Enteritidis de origen aviar y de humano con gastroenteritis en Ibagué, Colombia

Abstract Introduction: Salmonella Enteritidis is a major cause of human salmonellosis in the world, with contaminated eggs and raw chicken meat as the main routes of infection. The main Salmonella spp. serovars circulating in laying hen farms, the surface of eggs, and in raw chicken carcasses have been identified in Ibagué, Colombia. However, it is unknown whether those serovars are responsible for human gastroenteritis. Objective: To evaluate the genetic relationship between gastroenteritis and Salmonella Enteritidis isolates from poultry and humans using multilocus sequence typing (MLST). Materials and methods: Salmonella spp. was isolated from clinical cases of gastroenteritis (n=110). Antibiotic susceptibility tests, followed by serotyping and MLST were conducted and S. Enteritidis was compared to those from laying hen farms and marketed eggs. Results: Ten isolates of Salmonella spp. were obtained from the stools of people with gastroenteritis. The prevalence of Salmonella spp. in human stools was 9.09%, and S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Uganda (n=1), S. Grupensis (n=1), and S. Braenderup (n=1) were the main serotypes. MLST indicated that a common S. Enteritidis sequence type (ST11) was present in all three sources and showed the same antibiotic resistance pattern. Conclusion: Salmonella Enteritidis ST11 constitutes a link between consumption and manipulation of contaminated eggs and human gastroenteritis in Ibagué. Additional studies would be required to establish if other Salmonella serovars isolated from raw chicken meat are also associated with human gastroenteritis.

Resumen Introducción. Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo; los huevos contaminados y la carne de pollo cruda son sus principales fuentes de infección. En Ibagué, Colombia, se han identificado los principales serovares que circulan en granjas, superficies de huevos y canales de pollo, pero se desconoce si esos serovares son responsables de la gastroenteritis. Objetivo. Evaluar la relación genética entre los aislamientos de Salmonella Enteritidis de aves de corral y de humanos con la gastroenteritis mediante tipificación de multiloci de secuencias (Multilocus Sequence Typing, MLST). Materiales y métodos. Se aisló Salmonella spp. de casos clínicos de gastroenteritis (n=110). Se hizo la prueba de sensibilidad antibiótica, así como la serotipificación y la tipificación mediante MLST, y se comparó S. Enteritidis de humanos con la hallada en granjas de gallinas ponedoras y en huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp. a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9,09%, y se identificaron los serotipos S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup presentes en pacientes con gastroenteritis. Mediante la MLST, se comprobó que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y presentó el mismo patrón de resistencia antibiótica. Conclusión. Salmonella Enteritidis ST11 constituye un vínculo entre el consumo y la manipulación de huevos contaminados, y la gastroenteritis en humanos en Ibagué. Se requieren estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis en humanos.

Inactivation of phoPQ genes attenuates Salmonella Gallinarum biovar Gallinarum to susceptible chickens

Abstract Salmonella Gallinarum is a host-restrict pathogen that causes fowl typhoid, a severe systemic disease that is one of the major concerns to the poultry industry worldwide. When infecting the bird, SG makes use of evasion mechanisms to survive and to replicate within macrophages. In this context, phoPQ genes encode a two-component regulatory system (PhoPQ) that regulates virulence genes responsible for adaptation of Salmonella spp. to antimicrobial factors such as low pH, antimicrobial peptides and deprivation of bivalent cations. The role of the mentioned genes to SG remains to be investigated. In the present study a phoPQ-depleted SG strain (SG ΔphoPQ) was constructed and its virulence assessed in twenty-day-old laying hens susceptible to fowl typhoid. SG ΔphoPQ did cause neither clinical signs nor mortality in birds orally challenged, being non-pathogenic. Furthermore, this strain was not recovered from livers or spleens. On the other hand, chickens challenged subcutaneously with the mutant strain had discreet to moderate pathological changes and also low bacterial counts in liver and spleen tissues. These findings show that SG ΔphoPQ is attenuated to susceptible chickens and suggest that these genes are important during chicken infection by SG.

Isolation of Salmonella spp. in cattle egrets (Bubulcus ibis) from Fernando de Noronha Archipelago, Brazil

Abstract The growth of the population of cattle egrets (Bubulcus ibis) in the archipelago of Fernando de Noronha constitutes a threat to public health and biological diversity because of their competition with and predation on native species and the possibility of transmission of pathogens to human beings, livestock and native wildlife. The aim here was to search for, isolate and identify serovars of Salmonella in clinically healthy local cattle egrets. Cloacal swabs were obtained from 456 clinically healthy cattle egrets of both sexes and a variety of ages. The swabs were divided into 51 pools. Six of these (11.7%) presented four serovars of Salmonella enterica subspecies enterica: Salmonella serovar Typhimurium; Salmonella serovar Newport; Salmonella serovar Duisburg; and Salmonella serovar Zega. One sample was identified as S. enterica subspecies enterica O16:y:-. Results in this study suggest that cattle egrets may be reservoirs of this agent on Fernando de Noronha and represent a risk to public health and biological diversity.

Survey on pathogenic Escherichia coli and Salmonella spp. in captive cockatiels (Nymphicus hollandicus)

Abstract We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and Salmonella spp. Cloacal swabs were collected from 94 cockatiels kept in commercial breeders, private residencies and pet shops in the cities of São Paulo/SP and Niterói/RJ (Brazil). Three strains of E. coli from each individual were tested for the presence of ExPEC-, APEC- and DEC-related genes. We evaluated the blaTEM, blaSHV, blaOXA, blaCMY, blaCTX-M, tetA, tetB, aadA, aphA, strAB, sul1, sul2, sul3, qnrA, qnrD, qnrB, qnrS, oqxAB, aac (6)′-Ib-cr, qepA resistance genes and markers for plasmid incompatibility groups. Salmonella spp. was not detected. E. coli was isolated in 10% of the animals (9/94). Four APEC genes (ironN, ompT, iss and hlyF) were detected in two strains (2/27-7%), and iss (1/27-4%) in one isolate. The highest resistance rates were observed with amoxicillin (22/27-82%), ampicillin (21/27-79%), streptomycin (18/27-67%), tetracycline (11/27-41%). Multiresistance was verified in 59% (16/27) of the isolates. We detected strAB, bla TEM, tetA, tetB, aadA, aphaA, sul1, sul2, sul3 resistance genes and plasmid Inc groups in 20 (74%) of the strains. E. coli isolated from these cockatiels are of epidemiological importance, since these pets could transmit pathogenic and multiresistant microorganisms to humans and other animals.

Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken

ABSTRACT Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE ΔmotB) or non-flagellated (SE ΔfliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE ΔmotB and SE ΔfliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5 dpi). The SE ΔmotB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE ΔfliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.

Isolation of Escherichia coli and Salmonella spp. from free-ranging wild animals

Increasing interactions between humans, domestic animals and wildlife may result in inter-species transmission of infectious agents. To evaluate the presence of pathogenic E. coli and Salmonella spp. and to test the antimicrobial susceptibility of isolates, rectal swabs from 36 different free-ranging wild mammals were taken from two distinct natural sites in Brazil: Cantareira State Park (CSP, state of São Paulo) and Santa Isabel do Rio Negro Region (SIRNR, state of Amazonas). The swabs were randomly collected and processed for bacterial isolation, identification, characterization and antimicrobial resistance. Eighteen E. coli strains from CSP and 20 from SIRNR were recovered from 14 and 22 individuals, respectively. Strains from animals captured in CSP, the site with the greatest anthropization, exhibited a higher range and percentage of virulence genes, including an eae+/bfpA+ strain. Antimicrobial resistance was verified in strains originating from both sites; however, in strains from SIRNR, aminopenicillins were almost the exclusive antimicrobial class to which strains exhibited resistance, whereas in CSP there were strains resistant to cephalosporins, sulfonamide, aminoglycoside, tetracycline and fluoroquinolone, in addition to strains exhibiting multidrug resistance. Two strains of Salmonella enterica that are known to be associated with reptiles, serotypes Belem and 60:r:e,n,z15, were recovered only from Amazonian animals and showed susceptibility to all classes of antimicrobials that were tested. Although the potential impact of these pathogens on wildlife remains unknown, bacteria isolated from free-ranging wild animals may provide relevant information about environmental health and should therefore be more deeply studied.

A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species

Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil

The objective of this study was to identify mutations in the Quinolone Resistance Determining sources Regions (QRDR) of the gyrA, gyrB, parC, and parE genes and to determine if any of the qnr variants or the aac(6')-Ib-cr variant were present in strains of Salmonella spp. isolated in Brazil. A total of 126 Salmonella spp. strains from epidemic (n = 114) and poultry (n = 12) origin were evaluated. One hundred and twelve strains (88.8%) were resistant to nalidixic acid (NAL) and 29 (23.01%) showed a reduced susceptibility to ciprofloxacin (Cip). The mutations identified were substitutions limited to the QRDR of the gyrA gene in the codons for Serine 83, Aspartate 87 and Alanine 131. The sensitivity to NAL seems to be a good phenotypic indication of distinguishing mutated and nonmutated strains in the QRDR, however the double mutation in gyrA did not cause resistance to ciprofloxacin. The qnrA1 and qnrB19 genes were detected, respectively, in one epidemic strain of S. Enteritidis and one strain of S. Corvallis of poultry origin. Despite previous detection of qnr genes in Brazil, this is the first report of qnr gene detection in Salmonella, and also the first detection of qnrB19 gene in this country. The results alert for the continuous monitoring of quinolone resistance determinants in order to minimize the emergence and selection of Salmonella spp. strains showing reduced susceptibility or resistance to quinolones.

Prevalence of R-type ACSSuT in strains of Salmonella serovar Typhimurium DT193 isolated from human infections in Brazil / Prevalencia de resistencia de tipo ACSSuT en cepas de Salmonella serovariedad Typhimurium DT193 aisladas a partir de infecciones humanas en el Brasil

OBJECTIVE: To determine the prevalence of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (ACSSuT) in Salmonella serovar Typhimurium definitive [phage] type (DT) 193 strains isolated from human sources over the last four decades. METHODS: From 2008 to 2010, 553 DT193 isolates out of 810 human-origin Salmonella ser. Typhimurium phage-typed strains isolated from the 1970s through 2008 were selected and tested for ACSSuT resistance: 91 strains isolated during the 1970s, 65 from the 1980s, 70 from the 1990s, and 327 from 2000-2008. Resistance profiles were determined using the disk diffusion method. RESULTS: †An antimicrobial susceptibility assay indicated 20.9 percent, or 116, of all isolates tested were ACSSuT-resistant, 52.0 percent (287) were resistant to one or more drugs in the ACSSuT profile, and 27.1 percent (150) were nonresistant (susceptible to antimicrobials). Based on the assay, overall antimicrobial resistance was extremely high in the 1970s (affecting 99.0 percent of isolates from that period) and remained high during the 1980s, when 95.4 percent of isolates had some type of antimicrobial resistance and incidence of Salmonella ser. Typhimurium DT193 R-type ACSSuT increased to 73.8 percent. R-type ACSSuT dropped to 27.1 percent (19 isolates) during the 1990s, and to 5.2 percent (17) during 2000-2008, despite a substantial increase in the number of isolates tested (397 versus 204, 111, and 98, respectively, for the previous three decades). CONCLUSIONS: †Although prevalence of Salmonella ser. Typhimurium DT193 R-type ACSSuT in Brazil has decreased since the 1970s, ACSSuT resistance markers continue to circulate. Therefore, continuous surveillance should be conducted to evaluate the occurrence of Salmonella ser. Typhimurium DT193 and its antimicrobial resistance.

OBJETIVO: Determinar la prevalencia de resistencia a la ampicilina, el cloranfenicol, la estreptomicina, las sulfonamidas y las tetraciclinas (ACSSuT) en cepas de Salmonella serovariedad Typhimurium fagotipo definitivo (DT) 193 aisladas de fuentes de origen humano durante las cuatro últimas décadas. MÉTODOS: Entre el 2008 y el 2010 se seleccionaron 553 aislados de DT193 entre 810 cepas de Salmonella serovariedad Typhimurium fagotipificadas aisladas desde la década de 1970 hasta el 2008, y en ellos se analizó la resistencia a ACSSuT: se estudiaron 91 cepas aisladas durante la década de 1970, 65 aisladas durante la década de 1980, 70 aisladas durante la década de 1990, y 327 aisladas entre el 2000 y el 2008, respectivamente. Los perfiles de resistencia a los antibióticos se determinaron mediante el método de difusión en disco. RESULTADOS: El antibiograma indicó que 20,9 por ciento (116) de todos los aislados que se analizaron fueron resistentes a ACSSuT, 52,0 por ciento (287) fueron resistentes a uno o más antibióticos del grupo ACSSuT y 27,1 por ciento (150) no fueron resistentes (es decir, fueron sensibles a dichos antibióticos). Según el análisis, la resistencia general a los antibióticos fue muy alta en la década de 1970 (y comprendió a 99,0 por ciento de los aislados de ese período) y continuó siendo alta durante la década de 1980, cuando 95,4 por ciento de los aislados presentó algún tipo de resistencia a los antibióticos y la incidencia de Salmonella serovariedad Typhimurium DT193 con resistencia de tipo ACSSuT aumentó hasta 73,8 por ciento. La resistencia de tipo ACSSuT descendió a 27,1 por ciento (31 aislados) durante la década de 1990, y a 5,2 por ciento (17 aislados) entre el 2000 y el 2008, a pesar del aumento importante en el número de aislados que se evaluaron (397 frente a 204, 111 y 98 en las tres décadas anteriores, respectivamente). CONCLUSIONES: Aunque la prevalencia de Salmonella serovariedad Typhimurium DT193 con resistencia de tipo ACSSuT en el Brasil ha disminuido desde la década de 1970, los marcadores de resistencia a ACSSuT continúan en circulación. Por consiguiente, debe llevarse a cabo una vigilancia permanente para evaluar la aparición de infecciones por Salmonella serovariedad Typhimurium DT193 y su resistencia a los antibióticos.

Avaliação da reação em cadeia da polimerase e do isolamento bacteriológico convencional na detecção de Salmonella Dublin em amostras de fezes de bezerros infectados experimentalmente / Evaluation of polymerase chain reaction and standard microbiological techniques for detection of Salmonella Dublin in fecal samples of infected calves

The efficiency of the Polymerase Chain Reaction (PCR) combined with selective enrichment broth was compared with the standard microbiological techniques for detection of Salmonella Dublin in fecal samples of 10 to 15-days-old Holstein calves, experimentally infected with 10(8) CFU of Salmonella Dublin. Seventy-six fecal samples were analyzed using PCR associated with selenite cystine (SC) and Muller-Kauffmann tetrathionate (TMK) broths and standard microbiological techniques. Regardless of the selective enrichment broth used, the standard microbiological techniques were significantly better than PCR in detection of positive samples of Salmonella Dublin. However, the simultaneous use of both techniques provided detection of a larger number of positive samples. The SC broth was the best option as selective enrichment in both techniques.

Mechanism of infection of a human isolate Salmonella (3,10:r:-) in chicken ileum: ultrastructural study.

BACKGROUND & OBJECTIVE: Originally isolated from severe human food-poisoning cases, Salmonella (3,10:r:-), a monophasic variety of otherwise diphasic serotypes such as S. weltevreden and S. simi, causes serious infections in man, animals and poultry. Mechanism of infection of this versatile and deadly organism is important to understand for its control. The objective of this study was to enhance our understanding of infection of Salmonella (3,10:r:-) in vivo at cellular level. METHODS: Aliquots of 10(9) cfu of Salmonella (3,10:r:-) organisms were injected intra-ileally in 24 h pre-fasted 3 month old broiler chickens by standard ligated ileal loop method. After 18 h, the fluid accumulated in the ileum was drained and small tissue pieces were fixed in 2.5 per cent buffered (pH 7) glutaraldehyde and subsequently in 1 per cent aqueous osmium tetraoxide. Ultra-thin sections of araldite-embedded tissue pieces were examined under transmission electron microscope operated at 100 KV after staining with uranyl acetate and lead citrate. RESULTS: Over 70 per cent of salmonellae interacting within 300 nm with ileal epithelial cells developed numerous surface blebs of periplasmic extensions designated "periplasmic organelles" (POs). Large sized POs were apparently pinched off as outer membrane vesicles (OMVs), 50-90 nm in diameter. Type III secretion needle complex-like "rivet complexes" (RCs) were viewed to rivet the bacterial outer and inner membranes together, allowing only pockets of periplasm to expand/inflate in order to liberate OMVs. Many OMVs were found visibly docked on the plasma membrane of host epithelial cells. The invading organisms appeared to leave the epithelial cells so as to find entry into the lymphatic vessels, where, they again appeared to be closely interacting with ileal macrophages, by forming numerous POs and concomitantly liberating OMVs. Inside the cytoplasm of macrophages, numerous tight phagosomes were seen, each containing two organisms. The final stage appeared to contain replicated salmonellae, four in each loose phagosome and, at the same time, macrophages also showed signs of apoptotic disintegration, culminating in the release of replicated salmonellae. INTERPRETATION & CONCLUSION: Outer membrane vesicles released from a fiercely virulent human isolate, Salmonella 3,10:r:- pathogens have been implicated in translocating biochemical signals from the host-interactive organisms to the eukaryotic cells at both stages of invasion leading to epithelial cell and macrophage infection in vivo, in the chicken ileal model. A comprehensive cellular mechanism at ultrastructural level is outlined for typhoid-like Salmonella infections caused by this humans-infecting organism.

Occurrence of qacE/qacEDelta1 genes and their correlation with class 1 integrons in salmonella enterica isolates from poultry and swine.

In this study, a total of 122 Salmonella enterica isolates from poultry and swine were assessed for susceptibility to clinically important antibiotics and to benzalkonium chloride (BKC). All isolates were examined for the presence of the antiseptic resistance genes qacE and qacEDelta1 and intl1 (class 1 integrase). The intl1-positive strains were further investigated for the presence of the 3' conserved region. The results demonstrated widespread distribution of qacEDelta1 (27%) but no isolate with qacE was observed. The intl1 gene was identified in 23 isolates (70%) with qacEDelta1. All of the intl1-postive strains carried qacEDelta1 in 3' conserved segment, confirming that the qacEDelta1 gene is linked to the integrons. Increased MIC value to BKC was independent of the presence of qacEDelta1, and multiple antibiotic-resistant bacteria were no more tolerant to BKC than the non-multidrugresistant strains, regardless of the presence of qacEDelta1.

Distribution of virulence genes in Salmonella serovars isolated from man & animals.

BACKGROUND & OBJECTIVES: Pathogenesis of Salmonellosis depends upon a large number of factors controlled by an array of genes that synergise into the actual virulence of Salmonella. A study was undertaken to observe the distribution of three such genes, namely, Salmonella enterotoxin (stn), Salmonella Enteritidis fimbrial (sef and plasmid encoded fimbrial (pef genes, among different serovars of Salmonella enterica isolated from man and animals. METHODS: A total of 95 isolates belonging to S. Typhimurium (51), S. Enteritidis (36), S. Bareilly (3), and S. Paratyphi B (5) serovars were subjected to polymerase chain reaction (PCR) assay for the detection of stnl ssf and pef genes using their specific primers and the PCR products were analysed by 1 per cent agarose gel electrophoresis for the presence of the respective genes. RESULTS: Varying distribution pattern of these genes was observed amongst the isolates. While, stn was found in all the 95 strains, sef was found only among the S. Enteritidis isolates. The pef gene was found to be absent in 10 isolates including the three S. Bareilly isolates. INTERPRETATION & CONCLUSION: Findings indicated that the stn gene is widely distributed among Salmonella irrespective of the serovars and source of isolation. However, the sef gene appears to be serovar specific. Since the stn gene is found in all the isolates, it can be a viable target gene to explore the possibility of direct detection of Salmonella from samples from biological sources.

Biochemical Characteristics and Antimicrobials Susceptibility of Salmonella gallinarum Isolated in Korea

Fowl typhoid (FT) reported since 1992 in Korea is a septicemic disease of domestic birds caused by Salmonella gallinarum (S. gallinarum). The purpose of this study was to investigate the biochemical characteristics and antimicrobials susceptibility of field isolates of S. gallinarum isolated by year in Korea. A total of 258 isolates of S. gallinarum from 1995 to 2001 showed the same pattern in the majority of biochemical test such as IMViC (indole, methyl red, Voges-Proskauer and citrate utilization), carbohydrate fermentation and amino acid decarboxylation, and these results were almost in accordance with the traditional biochemical characteristics of S. gallinarum strain. When the antimicrobial susceptibility test against 258 isolates of S. gallinarum was performed by the disk diffusion method using 12 antimicrobial agents, all isolates from 1995 appeared to be susceptible to all of the antimicrobial agents tested except for tetracycline and oxytetracycline, whereas the vast majority of isolates from 2001 showed the reduced susceptibility to ampicillin (13.0%), gentamicin (43.4%), kanamycin (69.6%), enrofloxacin (6.5%), ciprofloxacin (10.9%), norfloxacin (52.5%) and ofloxacin (82. 6%). The prevalence of the prevalence of completely resisyany isolates resistant isolates to one or more drugs rapidly increased from 0% in 1995 to 93.5% in 2001. The minimal concentrations range of the majority of antimicrobial agents to inhibit 50% (MIC50s) against S. gallinarum isolates increased from 0.06 -- 8 microgram/ml in 1997 to 2 -- 256 microgram/ml in 2001. Especially, MIC50s for gentamicin and fluoroquinolones of isolates from 2001 increased over 10-fold than those of isolates from 1997. Therefore, our results indicate that sorbitol fermentation and arginine decarboxylation showed the diversity by isolates and the vast majority of isolates from 2001 showed the reduced susceptibility to antimicrobials tested.

Competitive exclusion against Salmonella gallinarum of Salmonella enteritidis infected chickens

To evaluate the degree of competitive exclusion against Salmonella gallinarum(S. gallinarum) of Salmonella enteritidis(S. enteritidis) infected chickens, fifty-six, 4-week old Hyline layer suspected of S. enteritidis infection were challenged with S. gallinarum. All chickens were tested for S. enteritidis isolation using cloacal swabs and serum plate agglutination test using S. enteritidis Ag. before challenge and classified into four groups(SE isolated, SE nonisolated, SE seropositive and SE seronegative). None of the SE isolated and the SE seropositive groups died after challenge and the average weight gains were 245.5g and 254.6g, respectively. But in the SE nonisolated and the SE seronegative groups, mortality was 18.2% and 20.6% and the average weight gains were 150.1g and 111.2g. The incidence of reisolation of S. gallinarum of the SE isolated and the SE seropositive groups were 41.7% and 47.6% from liver, 33.3% and 47.6% from spleen and 8.3% and 14.3% from cecum, respectively, and the SE nonisolated and the SE seronegative group were 63.6% and 64.7% from liver, 84.1% and 88.2% from spleen and 47.7% and 52.9% from cecum. The serological response of the SE isolated and the SE seropositive groups hardly changed from 75.0 and 81.8% before challenge to 75.0 and 85.7% after. But, the other two groups were found to be significantly higher after challenge and increased from 0 and 18.2% to 100%. Consequently, S. enteritidis preinfected chickens were found to be significant different in terms of mortality, weight gain, reisolation of S. gallinarum and serological response compared to noninfected chickens. Moreover, our study shows that S. enteritidis infected chickens appear strong competitive exclusion against the colonization of S. gallinarum.

Mucin allows survival of Salmonella typhi within mouse peritoneal macrophages

Salmonella typhi is a facultative intracellular human specific pathogen. Both immunocompetent and immunodeficient mice are resistant to S. typhi. However, when they are infected with S. typhi suspended in mucin, the bacteria become pathogenic and infect peritoneal phagocytic cells. The LD50 for mice was 10(5) bacteria suspended in 5 percent mucin; mouse survival was approximately 48 hours after injection. A high number of bacteria was recovered from peritoneal cells; transmission electron microscopy disclosed a large number of vesicles filled with S. typhi cells in peritoneal cells from infected animals. The addition of mucin to cultures of the reticuloendothelial cell line J774.3 also allowed invasion of the mammalian cells with S. typhi. These data indicate that mucin allows intracellular survival of S. typhi