Plague (Yersinia pestis)

John E. Bennett Dr. , in Mandell, Douglas, and Bennett's Principles and Exercise of Infectious Diseases , 2020

Microbiology

Y. pestis is an aerobic, gram-negative coccobacillus that exhibits bipolar staining with Giemsa, Wright, and Wayson stains. A member of the Enterobacteriaceae, it grows well on claret or MacConkey agar and in nutrient broths, such every bit brain-centre infusion. When incubated at 37°C, small colonies 1 to two mm in diameter are visible on claret or MacConkey agar after 24 to 48 hours. At 72 hours, colonies grown on blood agar tin take on a raised, irregular, "fried-egg" morphology. Y. pestis does not form spores and, unlikeY. enterocolitica andY. pseudotuberculosis, is nonmotile when incubated at lower temperatures. Information technology does not ferment lactose and is citrate, urease, and indole negative. 16

Genomic studies bespeak thatY. pestis evolved recently from the enteric pathogenY. pseudotuberculosis. 17 , 18 The transition from enteric to flea-borne pathogen required the ability to survive in the flea gut and to accomplish high concentrations in the blood of mammalian hosts. These traits were endowed in part through the acquisition of two plasmids encoding factors that are differentially expressed at temperatures encountered in fleas and mammals. 19 – 21 The 110-kilobase (kb) pMT1/pFra plasmid encodes bothYersinia murine toxin (Ymt), which is necessary for colonization of the flea midgut, and the fraction ane (F1) envelope antigen, which inhibits phagocytosis in mammals (Tabular array 229A.ane). The smaller nine.5-kb pPCP1 plasmid encodes a plasminogen activator protein (Pla protease) that is responsible for temperature-dependent coagulase and fibrinolysin activities. The origins of these plasmids remain uncertain; however, approximately half of the DNA sequences of the pMT1/pFra plasmid are shared with a plasmid ofSalmonella enterica serotype Typhi. 22

Equally with the other yersiniae, the plague bacillus also has an approximately 70-kb plasmid that mediates expression of virulence factors that prevent production of proinflammatory cytokines, increases resistance to phagocytosis, and enhances intracellular survival. 23 Chromosome-mediated factors include a stiff lipopolysaccharide endotoxin and a pigmentation factor, the hemin storage locus(hms), which regulates atomic number 26 uptake and enables the bacteria to form blockages of the flea gut that enhance transmission. 19 , 20

Y. pestis isolates tin can be classified into three chief biovars based on their ability to ferment glycerol and reduce nitrate. It has been postulated that these biovars—Antigua, Medievialis, and Orientalis—reflect strains associated with the first, second, and third pandemics, respectively. Various molecular methods propose, even so, that these biovars practice non correlate fully with phylogenetic relationships, and although Orientalis is associated with the third pandemic, the association of before pandemics with specific biovars remains uncertain. 18 , 24 , 25

Media for the Clinical Microbiology Laboratory

Audrey Wanger , ... Amitava Dasgupta , in Microbiology and Molecular Diagnosis in Pathology, 2017

Selective and Differential Media

MacConkey agar non only selects for Gram-negative organisms past inhibiting Gram-positive organisms and yeast but also differentiates the Gram-negative organisms by lactose fermentation. Lactose ferments will stain pink while the nonlactose ferments will be clear colonies. The media also has the added advantage of inhibiting the swarming of Proteus.

Eosin-methylene blue is some other media that electively grows Gram-negative organisms, while at the same time inhibiting Gram-positive orga­nisms and some yeast by making use of toxic dyes. Enteric bacteria will vary in appearance on this media depending on their ability to ferment lactose and sucrose fermentation. Escherichia coli will take a dark-green metallic sheen.

Additional media selective for Gram negatives but specifically designed to differentiate stool pathogens includes Hektoen enteric (HE) agar which contains bile salts. In improver to selecting for Gram-negative organisms the media also allows for the presumptive differentiation of Salmonella and Shigella from other Gram-negative organisms such as Eastward. coli. Escherichia coli and other lactose ferments will produce yellowish or orange colonies. Nonlactose fermenters including Shigella produce green colonies while Salmonella appears every bit black colonies due to production of hydrogen sulfide. Other organisms produce hydrogen sulfide and appear as black colonies on HE agar including Citrobacter spp.

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Salmonella Species

John E. Bennett Doctor , in Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases , 2020

Microbiology

Salmonellae are gram-negative, not–spore-forming, facultatively anaerobic bacilli that measure 2 to 3 past 0.4 to 0.half-dozen µm in size. Like other Enterobacteriaceae, they produce acid on glucose fermentation, reduce nitrates, and practice not produce cytochrome oxidase. 12 All organisms, exceptS. Gallinarum-Pullorum, are motile as a outcome of peritrichous flagella, and most practice not ferment lactose. However, approximately 1% of organisms can ferment lactose and therefore may non exist detected if only MacConkey agar or other semiselective media are used to identify Salmonella based on colorimetric assay for fermentation of lactose. The differential metabolism of sugars can exist used to distinguish manySalmonella serotypes; serotype Typhi is the just organism that does not produce gas on sugar fermentation. 12

Freshly passed stool is preferred for the isolation ofSalmonella and should exist plated directly onto agar plates. Low-selective media, such equally MacConkey agar and deoxycholate agar, and intermediate-selective media, such every bitSalmonella-Shigella, xylose-lysine-deoxycholate (Fig. 223.i), or Hektoen enteric agar, are widely used to screen for bothSalmonella andShigella spp. Selective chromogenic media, such as CHROMagarSalmonella (DRG International, Springfield, NJ), are more specific than other selective media, reduce the need for confirmatory testing and time to identification, and increasingly are used for the primary isolation and presumptive identification ofSalmonella from clinical stool specimens. thirteen Stool specimens can be directly inoculated into selenite enrichment goop before plating on master media to facilitate the recovery of low numbers of organisms. 13 HighlySalmonella-selective media, such every bit selenite with brilliant green, should exist reserved for use in stool cultures of suspected carriers and under special circumstances, such equally outbreaks. Bismuth sulfite agar, which contains an indicator of hydrogen sulfite product and does non contain lactose, is preferred for the isolation ofS. Typhi and tin be used for the detection of the 1% ofSalmonella strains (including mostSalmonella serogroup C strains) that ferment lactose. 14 Later master isolation, possibleSalmonella isolates tin can be tested in commercial identification systems or inoculated into screening media, such as triple-sugar–atomic number 26 and lysine-iron agar.

Directly detection of enteric pathogens from stool specimens past DNA-based syndrome panels is increasingly used by clinical laboratories to let providers to rapidly identify the cause of gastroenteritis. To ensure that outbreaks of similar organisms are detected and investigated, all specimens that test positive for nontyphoidalSalmonella (NTS) past culture-independent diagnostic testing and for which isolate submission is requested or required nether public wellness reporting rules should be cultured in the clinical laboratory or at a public health laboratory.

THE MICROBIOLOGICAL Examination OF FOODS

W.F. Harrigan , Margaret E. McCance , in Laboratory Methods in Microbiology, 1966

ane The detection and enumeration of indicator leaner

(a) Coliform organisms

MacConkey's broth and MacConkey'due south agar are not satisfactory media for the detection and enumeration of coliform organisms in foods. One of the almost reliable methods uses violet red bile agar in pour plate counts.

Procedure

Prepare duplicate sets of plates using 1 ml amounts of the chosen range of dilutions of the nutrient. Add to each plate fifteen ml of violet ruddy bile agar, melted and cooled to 45°C, mix well and allow to fix. Finally overlay with some other 5 ml of violet cherry bile agar. After allowing to solidify, invert the plates and incubate at 35°C for 24 hours. Subsequently incubation, count the number of dark red colonies. Normally typical coliform colonies will be 0·5 mm or more in diameter and show show of the atmospheric precipitation of bile salts in the medium immediately surrounding the colonies, but of form overcrowding of the colonies volition cause a reduction in size. In the case of foods containing carbohydrates other than lactose, media to which low dilutions of the nutrient have been added may requite anomalous results. With plates of ten−2 or lower dilutions it is therefore necessary to confirm presumptive coliform colonies either every bit coliforms by picking them off and inoculating lactose broth or MacConkey'southward broth and incubating at 35°C or equally East. coli past the Eijkman test (see page 94).

Culling procedure

If very modest numbers of coliforms are expected to be nowadays in a food sample, a dilution tube count may be carried out only it is recommended that lauryl tryptose broth be used rather than MacConkey'south broth. Pipette aseptically ane ml of each of the prepared dilutions of the food into each of five tubes of lauryl tryptose goop. Incubate at 35°C. Examine after 24 and 48 hours for the production of acid and gas. The production of acid is determined by the add-on of a pH indicator to the tubes subsequently incubation. A presumptive positive is recorded if a tube shows the production of acrid and either sufficient gas to fill up the concavity of the Durham tube or effervescence when the side of the test-tube is tapped. In routine testing gas production is usually regarded as indicating a presumptive positive without the demand for testing for acid product. Presumptive positive tubes at low dilutions (up to x−2) should exist confirmed by subculturing into lauryl tryptose broth and incubating at 35°C. For E. coli counts, subculture presumptive positive tubes into lauryl tryptose broth and incubate at 44 ± 0·25°C. The nearly probable number of coliforms or E. coli can exist calculated from probability tables (see Appendix).

(b) Enterococcis

The enterococcus is a useful indicator organism for some foods since, compared with E. coli, it is more resistant to freezing, low pH, and moderate heat treatment. Thus enterococci may often exist detected in frozen foods, fruit juices, or foods which have received a brief rut treatment, even when E. coli has been killed by the inimical conditions. Enterococci are here regarded as including Strep. faecalis and Strep. faecium (and the varietal forms Strep. faecalis var. liquefaciens, Strep. faecalis var. zymogenes) and Strep. durans but excluding Strep. bovis and Strep. equinus.

Procedure

Pipette 1 ml amounts of the chosen range of dilutions into a ready of Petri-dishes. Add to each plate fifteen ml of molten maltose azide tetrazolium agar at 45°C, mix thoroughly with the inoculum and permit to set. Incubate at 35°C for 48 hours. After incubation, count the number of colonies which are dark cerise or have a crimson or pink key area (if possible selecting plates with between 30 and 300 colonies). Summate and record the number of enterococci per gram of food.

Alternative procedure

If very small-scale numbers of enterococci are expected to be present in a food sample, a dilution tube count may be carried out, using maltose azide broth as the selective medium. Pipette aseptically one ml of each of the prepared dilutions into each of five tubes of maltose azide broth. Incubate at 35°C for 48 hours. Examine the tubes after incubation for acid product, which is indicative of enterococci. Calculate the near likely number of enterococci using probability tables (see Appendix).

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The Human Microbiome

Lee Goldman MD , in Goldman-Cecil Medicine , 2020

IDENTIFICATION METHODS | Chromogenic Agars

P. Druggan , C. Iversen , in Encyclopedia of Nutrient Microbiology (Second Edition), 2014

Fermentation Media

In traditional fermentation media, like MacConkey agar, the pH modify caused by fermentation of lactose is the outcome of a cascade of steps that starts with transportation of a sugar into the cell and through an additional 16 enzyme steps (run into Figure three) earlier acrid is generated and the colour of the medium changes.

Effigy 3. Enzymes involved in the fermentation of lactose to lactic acid. The lac operon is shown in blue. The Leloir pathway for conversion of galactose to glucose-6-phosphate is shown in red. The conversion of glucose to glucose-half-dozen-phosphate is shown in greenish. Mutual enzyme pathways are shown in black.

Including the command sequences for each gene, roughly 35 genetic elements could affect the production of acid in lactose fermenters. Assuming a phenotypic mutation rate of 5 × 10−8 and a population density of 1 × 10eight cfu ml−1 in a pure civilisation of a coliform, information technology can be estimated that around 1750 cells in the population with a single mutation might fail to ferment the sugar. This conservative approximate does not take into consideration other mutations that might touch the cells chapters to ferment sugars in general. Mutations in the lac operon and the common pathway are the virtually pregnant, but expression of inactive enzymes in the ii branches would halve the total product of acid by the cell. Depending on the buffering capacity of the medium, this may or may non cause false positives.

The large numbers of competitive microflora in food samples leads to a relatively big number of nonfermenting bacterial species. During development, media for Salmonella were designed to inhibit nonfermenting organisms as much every bit possible without inhibiting the target organism. The fermentable sugars were included to differentiate organisms that could not exist inhibited without inhibiting Salmonella. Mutants that interrupt the pathway shown in Figure 3 would announced as fake positives on media like Xylose lysine deoxycholate agar or brilliant green agar. Although both of these media include sucrose every bit an boosted fermentable carbohydrate, it can be seen from Figure three that the addition of a saccharide might improve the specificity of the medium, and on hydrolysis, the glucose and fructose would feed into the common pathway. If in that location is a mutation in the genetic elements for control and expression of the enzymes in this, information technology would impact the ability to generate acid from both sucrose and lactose.

The incidence of Salmonella in cooked food is estimated to be i.5%. The number of positives seen in a routine testing laboratory will depend in the cloth being tested and on the type of processing involved. From this brief discussion, we hope that we accept shown why false-positives colonies are a mutual feature of pathogen testing on traditional media. The implications of this volition be discussed later in this chapter.

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Overview of Bacteria

Audrey Wanger , ... Amitava Dasgupta , in Microbiology and Molecular Diagnosis in Pathology, 2017

Fastidious Gram-Negative Rods

Diverse fastidious Gram-negative rods are discussed in this section.

Haemophilus spp.

Haemophilus spp. do not grow on MacConkey agar. Haemophilus influenzae has been shown to cause invasive infection such equally meningitis and there is direct spread from h2o droplets from the upper respiratory tract of infected individuals. The evolution of the H. flu type B vaccine has decreased mortality. Haemophilus ducreyi causes chancroid which is a sexually transmitted disease that leads to a single genital ulcer. H. ducreyi usually occurs in Asia, Africa, and Latin America. Haemophilus parainfluenzae tin exist a colonizer of the upper respiratory tract and can cause sinusitis and bronchitis. H. influenzae do not abound on BAP and grow only on chocolate agar as they require X and V factors for growth. H. parainfluenza require only V factor and H. ducreyi but requires Ten gene. Haemophilus spp. do not produce oxidase. Product of beta-lactamase production should be tested for isolates. Cephalosporins and carbapenems can be used also every bit combination agents with a beta-lactamase inhibitor.

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Examination of faeces for bacterial pathogens

Southward.H. Gillespie MB, BCh, BAO, MRCP(UK), MRCPath , in Medical Microbiology Illustrated, 1994

Plesiomonas shigelloides

This organism gained its proper noun because it grows on MacConkey's agar with pale colonies and is agglutinated by Due south. sonnei antiserum with which it shares an antigen. Information technology is found in fresh and estuarine waters, and is more mutual in tropical areas. It is a cause of enterocolitis associated with the consumption of seafood, or ingestion of untreated h2o. A number of putative pathogenicity determinants take been reported including heat-labile and rut-stable enterotoxins. Plesiomonas shigelloides tin can be isolated on MacConkey's agar or DCA where most of these strains are not-lactose fermenters. It is oxidase positive and tin be identified using conventional biochemical techniques. It has similar susceptibilities to Aeromonas spp., although ampicillin sensitivity is more than common.

Examples of biochemical reactions of some vibrios are summarized in Tabular array 17.9.

Table 17.nine. Examples of biochemical reactions of vibrios

Species Colonies on TCBS VP Growth in food broth Indole Arabinose Cellobiose
0% with NaCl half dozen% 10%
V. cholerae Yellow +/– + +/– – + – –
V. parahaemolyticus Green – – +/– +/– + +/– –
V. mimicus Yellow – + +/– – + – –
V. vulnificus Green – – + – + – +
Aeromonas spp. Yellow/– +/- + – – + 5 v/–
P. shigelloides – – + – – + – –

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Exam of urine and pus

Southward.H. Gillespie MB, BCh, BAO, MRCP(United kingdom), MRCPath , in Medical Microbiology Illustrated, 1994

Culture

Routine screening skin swabs can be inoculated on claret and MacConkey's agar, which should be incubated aerobically. Additional media for the isolation of other pathogens, such every bit Sabouraud's and Lowenstein-Jensen are appropriate when fungal or mycobacterial infection is suspected. Similarly, Hoyle'south medium would be required where C. diphtheriae was suspected.

In swabs from burn sites for skin grafting, a selective media such equally crystal violet blood agar might exist used in add-on to heighten the recovery of betahaemolytic streptococci. Contempo work has indicated the importance of anaerobic species in cutaneous ulcers which form an of import office of the flora. Even so, the clinical utility of isolating and reporting these organisms is non clear.

A new species, Fusobacterium ulcerans, has recently been identified from cases of tropical ulcer.

When peel swabs are examined to identify carriers of multiresistant organisms, appropriate selective indicator media should be used. For the diagnosis of methicillin-resistant Southward. aureus (MRSA) carriers, skin swabs from multiple sites should exist placed in a salt-broth medium and subcultured onto nutrient agar containing four mg/1 methicillin. This means that the merely organisms to be identified are those able to tolerate high table salt and resistance to methicillin, predominantly methicillin-resistant S. aureus and some strains of coagulasenegative staphylcocci. Identification and susceptibility testing tin can and then definitively identify MRSA. It is essential that screening systems which may bring in big numbers of specimens are carefully designed to optimize the use of resources and maximize diagnostic yield.

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Shigella

C. Jenkins , in Encyclopedia of Food and Wellness, 2016

Detection

Shigella grows as non-lactose-fermenting colonies on MacConkey agar, although a more selective agar is used for the isolation of Shigella from feces, such equally deoxycholate citrate agar and xylose lysine deoxycholate agar. Biochemically, Shigella spp. are unable to ferment lactose or utilize citrate and urea or to produce lysine decarboxylase. They are nonmotile and produce acrid from glucose simply no gas. Biochemical identification is confirmed by serological reactions to specific Shigella antisera. Isolation of Shigella from food is difficult, as colonies can be obscured by more than robust commensal microbiota, such equally Escherichia, Enterobacter, and Proteus species, and the level of contamination can exist low.

More recently, a polymerase chain reaction (PCR) targeting the invasion plasmid antigen H (ipaH) gene has been used to detect the pathogen directly in fecal and food samples. This PCR approach is much more sensitive than conventional culturing. The ipaH cistron is the target of choice, as information technology is encoded multiple times on both the chromosome and the big virulence plasmid. Thus, the exam has increased sensitivity over PCRs targeting single copy genes, and detection of the pathogen is possible in the event the large virulence plasmid is lost.

At that place are a variety of Shigella PCR assays described in the literature. Most incorporate primers targeting the ipaH genes located on both the virulence plasmid and chromosome and an internal amplification control and have been well validated and tested on several loftier-risk foods. The specificity and sensitivity of the assays are approximately one–3 colony-forming units and v–l   fg of target (total) Deoxyribonucleic acid. Different mail service-enrichment extraction protocols accept been compared, and the InstaGene matrix was found to be the most consistent and sensitive.

Multiplex existent-time PCR assays for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), and Yersinia enterocolitica in fecal samples have been described in the literature. The sensitivity of the PCR is approximately 61 colony-forming units per ml for Shigella spp. The use of PCR in clinical settings increases the overall clinical sensitivity from 78% to 100% when compared with conventional culturing. The specificity was 99.4% for the PCR, compared with 99.9% for conventional culturing. The real-time method for the amplification of the ipaH gene in food as recommended by the European Union Reference Laboratory can exist accessed by following http://www.iss.it/binary/vtec/cont/EU_RL_VTEC_Method_07_Rev_0.pdf.

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