A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

BACKGROUND: To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. METHODS: We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. RESULTS: We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. CONCLUSION: The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

The MreB and Min cytoskeletal-like systems play independent roles in prokaryotic polar differentiation.

Establishment of an axis of cell polarity and differentiation of the cell poles are fundamental aspects of cellular development in many organisms. We compared the effects of two bacterial cytoskeletal-like systems, the MreB and MinCDE systems, on these processes in Escherichia coli. We report that the Min proteins are capable of establishing an axis of oscillation that is the initial step in establishment of polarity in spherical cells, in a process that is independent of the MreB cytoskeleton. In contrast, the MreB system is required for establishment of the rod shape of the cell and for polar targeting of other polar constituents, such as the Shigella virulence factor IcsA and the aspartate chemoreceptor Tar, in a process that is independent of the Min system. Thus, the two bacterial cytoskeletal-like systems act independently on different aspects of cell polarization.

Effect on bovine lactoferrin on the activation of the enteroinvasive bacterial type III secretion system.

Shigella and enteroinvasive Escherichia coli (EIEC) strains secrete virulence proteins by a complex machinery called the type III secretion (TTS) apparatus. Secretion of virulence proteins is a tightly-regulated phenomenon such that the TTS system is weakly active when bacteria are grown in common laboratory media. Activation of the TTS system is triggered by contact with eukaryotic cells, or can be artificially stimulated by the addition of Congo red dye to the growth medium. Exploiting the ability of bovine lactoferrin (bLf) to bind iron we have found that the TTS of EIEC strain HN280 seems to be activated in conditions of low-iron availability, obtained by incubation of bacteria with bLf enclosed within a dialysis bag. Activation of secretion was assessed by measuring the release of IpaB and C, chosen as reporters of secreted virulence proteins. The contribution of small bLf-derived components, diffusing across the dialysis membrane, in the release of Ipa proteins has also been determined. Activation of secretion was not due to bLf-induced damage of the HN280 outer membrane and was not associated with increased transcription of the mxi operon. Thus, low-iron availability might be an environmental signal perceived by enteroinvasive micro-organisms in order to modulate secretion of virulence proteins.

Escherichia coli in disguise: molecular origins of Shigella.

Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli. There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid. The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure.

Modelling and estimation of physical parameters in a sludge drying system.

In this paper is presented the study of a Sludge Drying System used to kill pathogenic organisms living in sludge. The system is modeled and the physical parameters thermal capacity, thermal resistance and thermal time constant are estimated using conventional estimation methods.

Correlación de la citología de moco fecal con el coprocultivo en niños con diarrea aguda / Correlation of fecal mucus citology with coproculture in children with acute diarrhea

Se realizó un estudio retrospectivo en el Hospital Infantil Privado de la Ciudad de México del 1 de enero al 31 de diciembre de 1997, con el objetivo de determinar la utilidad de la citología de moco fecal en el diagnóstico de diarrea aguda en niños y su relación con el germen identificado en el coprocultivo. Se incluyeron 170 niños con diagnóstico de diarrea aguda en niños y su relación con el germen identificado en el coprocultivo. Se incluyeron 170 niños con diagnóstico de diarrea aguda con una mediana de edad entre 12 y 120 meses. A todos los pacientes se les realizó citología de moco fecal y coprocultivo, encontrándose 11 casos de citología de moco fecal positivo (6.5 por ciento) y 159 negativos (87.6 por ciento); en el coprocultivo el agente etiológico se identificó en 21 casos (12.3 por ciento), de los cuales ocho correspondieron a E. coli (4.7 por ciento), ocho a Salmonella (4.7 por ciento) y cinco a Shigella (2.9 por ciento). La relación de la citología de moco fecal y el agente identificado en el coprocultivo no fue significativo, ya que mostró p=0.13 mediante la prueba de Fisher

[Procedure of standardization of Chlamydiae suspensions (author's transl)]. / Une méthode de standardisation des suspensions Chlamydiennes.

The enumeration of a chlamydiae suspension can be achieved by the addition of a standardized shigella suspension; the two cell populations, after coloration with acridine, are counted in microdrops. This procedure permits to plot a turbidity curve.

[Study of a collection of Shigella strains of provisional serovars (cultural and biochemical properties)]. / Izuchenie kollektsii shtammov shigell provizornykh serovarov (kul'tural'nye i biokhimicheskie svoistva).

The author studied 17 standard and local strains of shigellae of provisional serological variants (3873-50, 2000-53, 3341-53, 3615-53, 2710-54, 1621-54). By a number of biochemical signs (alkalization of citrate agar of Christiansen and Molke's lacmus, the growth on acetate medium, utilization of soluble starch) and resistance to the genus-specific dysentery bacteriophage the cultures of serological variant 2000-53 possessed no properties of shigellae and were escherichia according to the general characteristics. The enzymatic and cultural properties of the rest of provisional serological variants corresponded to the characteristics of bacteria belonging to Shigella genus.

Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen.

It is known that only certain strains of the family of Enterobacteriaceae, notably rough (R) mutants with the type R1 or R4 core, evoked antibodies in high titers against the common enterobacterial antigen (CA) after immunization of rabbits with heated cell suspensions. The present investigation deals with genetic and immunochemical aspects of certain R1 and R4 mutants isolated from Escherichia coli 08 and various Shigella serotypes which, unexpectedly, do not induce CA antibody formation. Immunochemical and genetical (transduction and conjugation) experiments revealed that the rough phenotype of these special mutants was evoked by a mutation of pyrE-linked rfa gene, called rfaL, which is involved in translocation of O-specific polysaccharides onto the lipopolysaccharide core. The transduction of the defective rfaL, allele into appropriate rough recipients results in transductants which have simultaneously lost the ability to evoke CA antibodies. This finding suggests that a close connection exists between the function of the rfaL gene and the expression of CA immunogenicity in R1 and R4 mutants. One of the strains synthesized neither O-hapten nor CA, suggesting a mutation in a region equivalent to the rfe genes of Salmonella.

Presence of rhapidosomes in various species of bacteria and their morphological characteristics.

Rod-shaped structures have been observed in cells of Pseudomonas, Photobacterium, Proteus, and Saprospira by use of the negative-contrast stain. These structures, referred to as rhapidosomes, appear to be normal components of these cells. Other bacteria including Escherichia, Salmonella, Shigella, Klebsiella, Micrococcus, Bacillus, Mycobacterium, Rhodospirillum, and Hydrogenomonas genera failed to reveal these structures. The rhapidosomes of Saprospira were found to consist of two rods, one encasing a narrower, longer structure. In contrast, the rhapidosomes of Pseudomonas, Proteus, and Photobacterium were without the rigid inner structure, but were occasionally seen filled with a homogeneous material as observed by the negative stain. Ultrathin sections of Pseudomonas cells indicate that these rhapidosomes are embedded within or are in close association with the nucleoplasm.