SALMAT cor : Microagglutination for Salmonella Flagella Serotyping

Abstract

Salmonella is a complex bacterial group with more than 2400 serovars widely distributed in nature; they are considered zoonotic because they can infect a variety of animals and be transmitted to humans. Usually, they cause alimentary acquired diseases such as gastroenteritis, typhoid fever, and others that can lead to severe complications and death. Serotyping is useful to differentiate among Salmonella, because it shows an important correlation with their clinical and epidemiological patterns; consequently, it is of high value for public health, animal health, agriculture, and industry. To characterize all known Kauffmann–White Salmonella serovars, over 250 antisera are required. Due to this and to high prices antisera, many laboratories worldwide have limitations in establishing Salmonella surveillance. Therefore, we developed and validated a Salmonella flagella microagglutination test (SALMATcor) that significantly reduces laboratory requirements of antisera. SALMATcor is based on scaling down, by fivefold, the antigen:antiserum volumes actually required for the reference method: flagella standard tube agglutination technique (STAT). Antigen preparation, temperatures, and incubation periods remained as established for STAT. The SALMATcor was validated according to ISO/DIS 16140:1999 protocol, which included 1187 comparisons of flagella determinations conducted by SALMATcor and STAT, on 141 Salmonella isolates of 12 common serotypes and the use of antiserum recommended for STAT. SALMATcor concordance was excellent (Cohen's kappa index 0.9982), obtaining relative accuracy >99.9% and relative specificity >99.9%. Additionally, SALMATcor has been used by CNRB-INCIENSA since 2004 to respond to all 40 Salmonella proficiency testing strains, provided by World Health Organization–Global Salmonella Surveillance Network, obtaining 100% concordance on serovar identification. On the basis of the results achieved with SALMATcor and considering that it also significantly reduces antiserum expenses, hand labor, glassware, and bench top and water bath space requirements (microtiter plates and micropipette tips are the only additional supplies), we envision that SALMATcor will contribute to establish a sustainable Salmonella serovar surveillance worldwide.

Introduction

S almonella genus is a complex bacterial group widely distributed in nature. They are often isolated from the intestinal tract of warm- and cold-blooded animals, as well as from environmental samples contaminated with fecal discharges (Brenner et al., 2000). The genus Salmonella includes pathogens affecting humans and animals, and the vast majority is considered zoonotic (WHO, 2005). In humans, Salmonella serotypes such as Typhi can cause typhoid fever, a life-threatening disease. However, most Salmonella strains typically cause gastroenteritis in healthy persons and can cause a severe illness in the young, the elderly, and patients with weakened immunity. Salmonella spp. have proved to be of great concern for public health, animal health, agriculture, and food industry (WHO, 2005; Coburn et al., 2007).

For taxonomic purposes, this genus has been subdivided in two species: Salmonella enterica (including six subspecies) and Salmonella bongori (Baron et al., 1995). Serological characterization of these bacteria, according to Kauffmann–White taxonomic scheme, has provided a useful tool for differentiation among isolates, and proved to be a great asset to correlate with their clinical and epidemiological patterns. This scheme is based on a combination of biochemical reactions and serotyping of the somatic “O,” flagellar “H,” and capsular “Vi” antigens (Ewing, 1986). Among these more than 59 somatic antigens, 87 flagella antigens (FAs) have been recognized, and over 2400 serotypes are described for Salmonella (Grimont and Weill, 2007). To be able to characterize such a diverse group of microorganisms, most Latin American laboratories use a standard tube agglutination technique (STAT) for flagella serotyping, whereas European laboratories usually work with slide agglutination. Both these methods require more than 250 different well-characterized antisera (Cai et al., 2005), and these are very expensive for developing countries. Therefore, notwithstanding the support of international organizations like World Health Organization–Global Salmonella Surveillance Network (WHO-GSS), now WHO Global Foodborne Infections Network (WHO-GFN), to reinforce the ability of local and regional laboratories for Salmonella characterization, this goal has not been completely achieved, at least in the Central America Region, due to economic limitations for acquiring antisera.

During the late 1970s Ship and Rowe developed a mechanized technique for Salmonella serotyping that uses small volumes of commercial antisera (25 μL). However, it depended on the purchase of specialized equipment. To this point, other typing approaches are currently being developed, which also require acquisition of specialized equipment as well as expensive reagents like polymerase chain reaction (PCR), protein microarrays, and Luminex (Agron et al., 2001; Herrera-León et al., 2004; McQuiston et al., 2004; Cai et al., 2005).

In our laboratory due to budget limitations and to the importance of maintaining Salmonella-laboratory-based surveillance, we developed a manual microtiter flagella test that has been used regularly for Salmonella serotyping since 2004. This microtest requires smaller amounts of reagents than STAT and has allowed us to maintain Salmonella surveillance programs in our country. Therefore, the aims of this work are to describe the so-called Salmonella microagglutination technique (SALMATcor) and to present the validation results with STAT as a reference method.

Materials and Methods

The SALMATcor was developed and validated at the “Centro Nacional de Referencia en Bacteriología from the Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud” (CNRB-INCIENSA). The performance of this technique was compared with STAT as a reference method (Caffer et al., 2008).

Salmonella serovar determination according to Kauffmann–White requires a combination of analyses such as specific biochemical tests, the determination of the O, Vi, phase 1, and phase 2 FAs, including the phase inversion procedure when required. In this work, those tests, but FA determinations, were conducted as previously described (Ewing, 1986; Caffer et al., 2008). All antisera used in the two methods were recommended for STAT application and were diluted according to the manufacturer's technical specifications. To validate SALMATcor, Salmonella isolates were analyzed by both methods as follows.

The STAT for Salmonella flagella serotyping was performed as described by Ewing (1986) and Caffer et al. (2008). Briefly, for FA preparation each Salmonella strain was inoculated on brain heart infusion agar from Becton Dickinson and Company (BD; Franklin Lakes, NJ) and incubated overnight at 36°C ± 1°C, and then subcultured in 5 mL of a flagella broth (15 g tripticase broth, 13 g triptose broth per liter of distillated water) for 18–24 h at 36°C ± 1°C without agitation. Then, 0.5 mL of the culture was separated and stored at 4°C as a backup. To the remaining 4.5 mL of FA, an equal volume of 0.85% (w/v) saline containing 1% (v/v) of formalin was added and incubated at room temperature for at least 1 h to obtain the formalinized FA (FFA).

For each antigen to be determined, 500 μL of FFA was placed in a 13 × 100 mm test tube and 100 μL of diluted monovalent antiserum was added (or 500 μL if using polyvalent antiserum). Then, the tube was incubated in a water bath without agitation for 1 h at 50°C ± 1°C. The tube was removed from the water bath avoiding abrupt movements that could disrupt the aggregates, and was examined for agglutination under indirect white light. The result was recorded and interpreted according to the correspondent scheme. For each strain an autoagglutination control test was also set. For this, 500 μL of FFA was placed into a 13 × 100 mm test tube and 100 μL of 0.85% saline was added. This tube was incubated as the sample and was also examined to confirm the lack of autoagglutination, before interpreting the sample results. If autoagglutination occurred, the test cannot be interpreted and the strain should be reanalyzed.

The SALMATcor for Salmonella flagella serotyping was performed as follows. The FFA preparation for each Salmonella strain to be tested was prepared like it was prepared for STAT. For each antigen to be evaluated, 100 μL of FFA was placed into a well of a 96-well polystyrene microplate (round or flat bottom) and 20 μL of diluted monovalent antiserum was added (or 100 μL if using polyvalent antiserum). The plate was covered with a lid and then incubated in a water bath over a floating platform for 1 h at 50°C ± 1°C. After incubation, to avoid abrupt movements that could disrupt the aggregates, the wells were examined for agglutination under indirect white light, interpreting positive and negative reactions as shown in Figure 1. As it required for STAT, an autoagglutination control is always required. In this case, in one well of the plate 20 μL of saline was added to 100 μL of FFA, and the results were recorded and interpreted.

FIG. 1.

Agglutination reactions as seen in SALMATcor. In the middle well, a positive agglutination reaction can be observed, flanked by two negative reactions.

Validation procedure

For the intralaboratory validation, 141 Salmonella strains of 12 different serovars isolated from humans, animals, food, and environment were used (Table 1). In total, 1187 Salmonella H antigens were characterized by each method to compare concordance between STAT and SALMATcor. For the H antigen characterization, the following were used: BD polyvalent H Salmonella antisera; Spicer-Edwards E1, E2, E3, and E4; 1 Complex, EN Complex, L Complex, and G Complex; and b, e, h, r, v, z15, m, q, and 5, among other monovalent H Salmonella antisera from different brands—BD, Denka Seiken (1990), and Malbrán (INPB-ANLIS Instituto Dr. Carlos G. Malbrán, Buenos Aires, Argentina). All of these were recommended for STAT application. For the intralaboratory statistical validation of the assay, the relative accuracy (AC), relative specificity (SP), and relative sensibility (SE) of the SALMATcor compared to the STAT and the lower confidence level for each performance indicator (at 95% of confidence) were determined as described in ISO/DIS 16140:1999 “Microbiology of Food and Animal Feeding Stuffs—Protocol for the Validation of Alternative Methods” (1999). AC was defined as the degree of correspondence between the response obtained by the STAT and the response obtained by the SALMATcor. The SE was defined as the ability of the SALMATcor to detect the antigen when it is detected by the STAT. The SP was defined as the ability of the SALMATcor not to detect the antigen when it is not detected by the STAT. The Cohen's kappa index was used to estimate the concordance between the results obtained by both analytical methods (Landis and Koch, 1977).

Table 1.

Characteristics of the Salmonella Isolates and Selected Flagella Antigens Used for SALMATcor Intralaboratory Evaluation

Serovar	No. of isolates	Origin (no. of isolates)	Flagella antigens ^a
Brandenburg	4		l,v:	e,n,z₁₅
Braenderup	1	Human	e,h:	e,n,z₁₅
Enteritidis	31	Human (29), food (2)	g,m:	—
Heidelberg	20	Human (2), food (18)	r:	1,2
Infantis	8	Human (4), food (4)	r:	1,5
Litchfield	1	Food	l,v:	1,2
Newport	6	Human (5), food (1)	e,h:	1,2
Pakistan	1	Human	l,v:	1,2
Panama	19	Human	l,v:	1,5
Paratyphi B	29	Food	b:	1,2
Saintpaul	7	Human (4), food (1), animal (2)	e,h:	1,2
Sandiego	14	Human (12), food (1), environment (1)	e,h:	e,n,z₁₅
Total	141	Human (77), food (61), animal (2), environment (1)

A total of 1187 antigen determinations were analyzed using the 141 different isolates.

Additionally, serovar identification results obtained by CNRB-INCIENSA using SALMATcor to analyze the 40 Salmonella isolates sent by WHO-GSS External Quality Assurance Program (EQAS), from 2004 to 2009 (Table 2), were used to determine the concordance with the expected serovar results established by Danish Veterinary Laboratory, National Food Institute of Denmark (DVI), a WHO collaborating center that uses slide agglutination technique for Salmonella flagella serotyping (www.who.int/salmsurv/activities/GSS_EQAS/en/).

Table 2.

External Quality Assurance Program Isolates Analyzed by SALMATcor That Are Concordant with the World Health Organization–Global Salmonella Surveillance External Quality Assurance Program 2004–2009 ^a

Expected serovar	No. of isolates	Year of EQAs	Expected flagellar antigenic formula
Albany	1	EQAS-2009	z₄,z₂₄:	—
Blockley	1	EQAS-2008	k:	1,5
Braenderup	1	EQAS-2004	e,h:	e,n,z₁₅
Brandenburg	1	EQAS-2009	l,v:	e,n,z₁₅
Bredeney	1	EQAS-2009	l,v:	1,7
Chester	1	EQAS-2004	e,h:	e,n,x
Concord	1	EQAS-2007	l,v:	1,2
Corvallis	2	EQAS-2004	z₄,z₂₃:	—
Elisabethville	1	EQAS-2007	r:	1,7
Enteritidis	5	EQAS-2004, 2006, 2007, 2008, 2009	g,m:	—
Give	2	EQAS-2004, 2006	l,v:	1,7
Heidelberg	1	EQAS-2004	r:	1,2
Hiduddify	1	EQAS-2008	l,z₁₃,z₂₈:	1,5
I 1,4,(5),12:i:-	1	EQAS-2006	i:	—
Indiana	1	EQAS-2008	z:	1,7
Isangi	1	EQAS-2007	d:	1,5
Javiana	1	EQAS-2008	l,z₂₈	1,5
Livingstone	1	EQAS-2007	d:	l,w
London	1	EQAS-2006	l,v:	1,6
Mbandaka	2	EQAS-2004, 2007	z₁₀:	e,n,z₁₅
Meleagridis	1	EQAS-2008	e,h:	l.w
Montevideo	1	EQAS-2007	g,m,s:	—
Muenster	1	EQAS-2009	e,h:	1,5
Oranienburg	1	EQAS-2008	m,t:	—
Poona	1	EQAS-2007	z:	1,6
Reading	1	EQAS-2006	e,h:	1,5
Rissen	1	EQAS-2006	f,g:	—
Saintpaul	1	EQAS-2006	e,h:	1,2
Sandiego	1	EQAS-2009	e,h:	e,n,z₁₅
Stanley	1	EQAS-2009	d:	1,2
Thompson	1	EQAS-2008	k:	1,5
Virchow	1	EQAS-2006	r:	1,2
Worthington	1	EQAS-2009	z:	l,w
Total	40	EQAS-2004–2009

There were no EQAS in 2005.

EQAS, External Quality Assurance Program.

Results

The present work describes and validates a new Salmonella flagella serotyping method in a microtiter plate format that reduces the STAT antisera requirements by fivefold.

For the intralaboratory validation, the outcome of each of 1187 antigen–antisera reactions was analyzed by SALMATcor and STAT, resulting in a total of 460 positive agreements, 726 negative agreements, 1 false-negative reaction, and no false-positive reaction. Performance indicators were 99.92% AC, 100% SP, and 99.78% SE. The lower confidence limit was >99%. The concordance between the SALMATcor and the STAT gave a Cohen's kappa index = 0.9982. The only mismatch observed was a negative reaction instead of a positive one, which was later confirmed as an error in the reference method (STAT).

The characterization of the 40 EQAS strains with SALMATcor gave 100% concordance with the expected serovars reported by DVI (Table 2).

Discussion

Concordance between SALMATcor and STAT was demonstrated by the intralaboratory validation assay. The 100% serovar concordance results with EQAS strains strongly suggest also a good correlation with other methods such as slide agglutination.

Not only does the SALMATcor technique, as described in this article, allow for a fivefold reduction of Salmonella H antisera expenses, but also, like most microtiter adaptations, it is easier to perform, allowing technicians to handle larger amounts of samples at the same time, and requires less glassware, water bath, and bench top space.

Nowadays, developed countries are moving toward newer Salmonella typing technologies such as PCR, Luminex, or microarrays (Cai et al., 2005). Although these methods show promising results and have advantages, they are still not ready for complete substitution of the conventional agglutination methods. So far, these new approaches have detected only a limited number of serovars, and many different genetic markers are still to be developed or verified for numerous serovars (Agron et al., 2001; Herrera-Leon et al., 2004; McQuinston et al., 2004). On the other hand, for developing countries, these technologies remain expensive; therefore, many laboratories worldwide will continue relying on serological characterization to maintain Salmonella surveillance programs. For those laboratories that already perform Salmonella serotyping using STAT, adopting SALMATcor is recommended, because it reduces the H antiserum expenses significantly, and does not require new equipment unlike Shipp and Rowe's (1980), PCR, microarray, and Luminex methods do. Further, with SALMATcor, laboratories do not require changes in their current serotyping algorithm or in the type or source of antisera, or do not have to alter the manufacturer-recommended antisera dilutions. All these conditions can be maintained, because the antigen:antiserum volume ratio of STAT is conserved. The microtiter format also allows for easy and quick visual agglutination readings, as shown in Figure 1. The only new requirements to set up this technique are the round- or flat-bottomed microtiter plates and micropipettes tips, which are materials often available in public health laboratories.

Laboratories working with STAT or SALMATcor must be aware that, as with all other serological methods, nontypable strains may be encountered. Problems with those strains should be overcome the same way as with STAT and other serological techniques. Strains that autoagglutinate cannot be serotyped unless autoagglutinating characteristics are reverted by serial subculture procedures.

In other regions, aside from Latin America, Salmonella flagella characterization may be performed by slide agglutination techniques. However, it should be taken into consideration that the antisera required for this method are different, with higher antibody titers from those recommended for the STAT, and are not meant to be used in a tube agglutination assay. Although we did not perform slide agglutination in our laboratory, the comparison of results obtained by SALMATcor resulted in 100% concordance with the expected serovar identification established for EQAS WHO-GSS program.

Conclusions

An easy alternative procedure that reduces laboratory expenses and makes Salmonella serovar surveillance more accessible is described. This new method is as accurate and specific as the STAT, showing an excellent concordance in the intralaboratory validation assay and the WHO-GSS EQAS program.

This microagglutination technique can be adopted even by low-income laboratories in developing countries, independently of antisera's brand/source, as long as they are designed for STAT applications. For those laboratories that already perform Salmonella serotyping by the tube technique or that are planning to implement it, adopting the SALMATcor is recommended. Therefore, we consider that SALMATcor will contribute to improve Salmonella surveillance worldwide.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Agron

, Walker

, Kinde

, Sawyer

, Hayes

, Wollard

, Andersen

. Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar Enteritidis. Appl Environ Microbiol, 2001; 67:4984–4991.

Baron

, Weissfeld

, Fuselier

, Brenner

. Classification and identification of bacteria. Manual of clinical microbiology, 6th Murray

, Baron

, Pfaller

, Tenover

, Yolken

. Washington, DC: American Society for Microbiology, 1995; 249–265.

Brenner

, Villar

, Angulo

, Tauxe

, Swaminathan

. Salmonella Nomenclature. J Clin Microbiol, 2000; 38:2465–2467.

Caffer

, Terragano

, Binsztein

. “Manual de procedimientos: Diagnostico y caracterización de Salmonella spp.” Buenos Aires, Argentina: WHO Global Salmonella Surveillance Network. Regional Reference Center for South America, Instituto Nacional de Enfermedades Infecciosas A.N.L.I.S. “Dr. Carlos G. Malbran,” 2008; 49–52.

Cai

, Lu

, Muckle

, Prescott

, Chen

. Development of a novel protein microarray method for serotyping Salmonella enteric strains. J Clin Microbiol, 2005; 43:3427–3430.

Coburn

, Grassi

, Finlay

. Salmonella, the host and disease: a brief review. Immunol Cell Biol, 2007; 85:112–118.

Denka Seiken Co., Ltd. Salmonella Antisera “Seiken” in Vitro diagnostic reagent ref. (62E) No. 1438. Bacteriology product information. Tokyo: Denka Seiken Co., Ltd., 1990; 9–12.

Ewing

. The genus Salmonella. Identification of Enterobacteriaceae, 4th Edwards

, Ewing

. New York: Elsevier Science Publishing Co., 1986; 181–318.

Grimont

, Weill

. Antigenic formulas of the Salmonella serovars. 9th. Paris, France: WHO Collaborating Centre for Reference and Research on Salmonella, Institute Pasteur, 2007.

10.

Herrera-León

, McQuiston

, Usera

, Fields

, Garaizar

, Echeita

. Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp. J Clin Microbiol, 2004; 42:2581–2586.

11.

[ISO] International Standard Organization. Microbiology of food and animal feeding stuffs: Protocol for the validation of alternative methods. International Standard ISO/DIS 16140:1999. Brussels: ISO, 1999; 5–12.

12.

Landis

, Koch

. The measurement of observer agreement for categorical data. Biometrics, 1977; 33:159–174.

13.

McQuiston

, Parrenas

, Ortiz-Rivera

, Gheesling

, Brenner

, Fields

. Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella. J Clin Microbiol, 2004; 42:1923–1932.

14.

Shipp

, Rowe

. A mechanized microtechnique for Salmonella serotyping. J Clin Path, 1980; 33:595–597.

15.

[WHO] World Health Organization. Drug-resistant Salmonella, Fact Sheet N°139. [homepage on the Internet]. Geneva: WHO. 2005. www.who.int/mediacentre/factsheets/fs139/en/updated 2005 April; cited 2009 Jun 30.