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Valeriana officinalis L. (Valerianaceae). The name derives from the Latin word valere (to be in health). Related subspecies include V. exalata, V nitida, V. edulis, and V. wallichii, commonly called Valerian, or sometimes Valeriana. In French, it is Valériane officinale or Herbe aux chats; in German, Baldrianwurzel or Baidrian.
Valerian will grow in any temperate climate. Dozens of different subspecies and varieties are recognized. Valerian's active ingredients are found in a volatile oil extracted from its roots. The oil contains a chemical called bornyl acetate, along with smaller amounts of valerenic acid, acetoxyvalerenic acid and valerenal, collectively known as valepotriates. A group of related molecules with sedative properties, called sesquiterpenes, are also found in the oil. The essential oil content varies widely from subspecies to subspecies. Concentrations of bornyl acetate can vary from less than one percent to more than 33 percent, depending on the variety of plant. The three valepotriates, the compounds considered most likely to be responsible for Valerian's sedative effects, are found together only in V. Officinalis.
Herbal drug manufacturers usually adjust their products to contain a standardized amount of the valepotriates, and then set the price accordingly. Commercial products made with V. Edulis are common, but they do not contain all three valepotriates or any sesquiterpenes. They may, therefore, be less effective.
Use for restlessness, sleeping disorders based upon nervous complaints.
Valerian is a mild, sleep-inducing, sedative. Studies designed to prove these claims scientifically are few and far between, but the few studies that have been done support that notion. The largest of the sleep studies involved 128 volunteers with insomnia. Each took 400 mg of valerian extract, or placebo, 30 minutes before going to bed. The time required to fall asleep, and the number of times participants woke during the night, was reduced. The improvement was most marked in women, and in people under the age of 40. Other controlled studies have produced similar results. The problem is, there have been so few studies done with valerian.
Cavadas C, Araujo I, Cotrim MD, Amaral T, Cunha AP, Macedo T, et al. In vitro study on the interaction of Valeriana officinalis L. extracts and their amino acids on GABAA receptor in rat brain. Arzneimittelforschung 1 995;45 (7): 753-5.
Cott J. NCDEU update. Natural product formulations available in Europe for psychotropic indications. PsychPhrmacol Bull 1995;3 1(4): 745-51.
Kammerer E. [Phytogenic sedatives-hypnotics-does a combination of valerian and hops have a value in the modern drug repertoire?]. Z Arztl Fortbild (Jena) 1993;87(5):401-6.
Leuschner J, Muller J, Rudmann M. Characterisation of the central nervous depressant activity of a commercially available valerian root extract. Arzneimittelforschung 1 993;43(6) :638-41.
Lindahl 0, Lindwall L. Double-blind study of a valerian preparation. Pharmacol Biochem Behav 1989;32(4): 1065-6.
Wagner J, Wagner ML, Hening WA. Beyond benzodiazepines: alternative pharmacologic agents for the potential to help support symptoms of insomnia. Ann Pharmacother 1998;32(6):680-91.
There are very few concerns with valerian use. The United States Pharmacopeia, which cautions against the use of any botanical product for more than two weeks, admits that in the case of valerian, there is a "lack of reported harmful side effects." Even massive overdoses seem to produce little toxicity. In Hong Kong, where valerian products are extremely popular and widely available, and where suicide attempts with valerian are not uncommon, not much happens. With appropriate medical treatment, almost all cases recover without any residual effects. Users do not need to be concerned about valerian's nasty smell. It does not mean that the valerian has gone bad; only that one of the components of valerian oil develops an unpleasant aroma with storage.
Buyers need to beware of what they are purchasing. Sesquiterpene content in commercial preparations can vary by a factor of ten, and if the product is made just from V. Edulis, the sesquiterpene content is usually nil. Since no one is quite sure what part of the oil produces the positive effects, buying a product missing one of valerian's active ingredients does not seem to make much sense.
Manufacturers in the United States are not required to provide any data on the purity of their herbal products, and content may vary considerably from batch to batch, and from manufacturer to manufacturer. Make sure you are dealing with a reputable manufacturer. Also, don't expect to get much benefit from valerian teas. Valerian's active ingredients are found in the oil, and do not dissolve in water. Finally, valerian is a sedative. That means it should not be combined with other sedatives, and definitely should not be taken with alcohol.
None of the molecules present in valerian is known to cause any interference with workplace drug tests. But sedative drugs can cause impairment. If you take valerian, and then drive, and are stopped by the police, you can still be charged with "driving under the influence" (DUI), even though you have not been drinking alcohol. Nor is using valerian tincture a good excuse for flunking a Breathalyzer test. Valerian tinctures do contain alcohol, but not enough alcohol to raise blood levels above legal limits, even if you use many times the recommended dose; that argument has already been tried in court, and it does not work.

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Part I of this article discussed the expressed justifications for applying the "broadest reasonable interpretation" claim construction standard to new claims submitted in patent applications to the U.S. Patent and Trademark Office, and the claim construction standard established in Phillips v. AWH Corp., 415 F.3d 1303 (Fed. Cir. 2005), to granted claims in district court. Part II will discuss the potential implications of the PTO's recent rule change, under which the Phillips standard will now be applied to substitute claims proposed in a motion to amend during inter partes review at the Patent Trial and Appeal Board instead of the broadest reasonable interpretation standard.
In an effort to create "greater consistency and harmonization," the PTO issued a final rule change on Oct. 11 to revise the rules for inter partes review, post-grant review and covered business method proceedings "by replacing the BRI standard for interpreting unexpired patent claims and substitute claims proposed in a motion to amend with the same claim construction standard that would be used to construe the claim in a civil action" (emphasis added). This creates a potential new source of disharmony regarding the examination of new patent claims: While patent examiners at the PTO will construe new claims submitted in patent applications using the broadest reasonable interpretation standard, the PTAB will construe new "substitute claims" submitted in motions to amend using the narrower Phillips standard. This appears to contradict the U.S. Court of Appeals for the Federal Circuit's justifications described in Part I of this article for applying the broadest reasonable interpretation standard to new claims, while reserving the narrower Phillips standard for claims that have already been granted and thus enjoy a presumption of validity. Indeed, the Federal Circuit noted in In re Morris, 127 F.3d 1048 (Fed. Cir. 1997), that applying the narrower district court standard to new claims would be contrary to the justifications for applying each of these standards in their respective forums. There, the Federal Circuit explained that "[i]t would be inconsistent with the role assigned to the PTO in issuing a patent to require it to interpret claims in the same manner as judges who, post-issuance, operate under the assumption the patent is valid. The process of patent prosecution is an interactive one. ... This promotes the development of the written record before the PTO that provides the requisite written notice to the public as to what the applicant claims as the invention. ... [P]ublic notice is an important objective of patent prosecution." 127 F.3d at 1054; see also In re Yamamoto, 740 F.2d at 1572 ("The PTO broadly interprets claims during examination of a patent application ... This approach serves the public interest," whereas "[d]istrict courts may find it necessary to interpret claims to protect only that which constitutes patentable subject matter to do justice between the parties."). Thus, according to the Federal Circuit's directives, by changing the claim construction standard applied in the PTAB to new "substitute claims" to the narrower Phillips standard, the PTO may be depriving the public of the protection from overly broad, poorly defined, and possibly non-novel patent claims that used to be provided by the broadest reasonable interpretation standard.
Moreover, the fact that an inter partes review, in which new "substitute claims" can be proposed in a motion to amend, is a post-grant procedure, while an examiner's review of new claims in a patent application is a pre-grant procedure, does not appear to justify this potential disharmony in claim construction standards. To the contrary, in post-grant procedures such as reissues and reexaminations, the PTO applies the broadest reasonable interpretation claim construction standard. In re Cuozzo Speed Techs., LLC, 793 F.3d 1268, 1276 (Fed. Cir. 2015), aff'd sub nom. Cuozzo Speed Techs., LLC v. Lee, 136 S. Ct. 2131 (2016) ("This court has approved of the broadest reasonable interpretation standard in a variety of proceedings, including ... post-grant proceedings such as reissues and reexaminations."). This remains unchanged by the PTO's new rules. Accordingly, the rule change's application of the Phillips standard to substitute claims submitted in motions to amend may be inconsistent not only with the Federal Circuit's directives regarding construction of new claims, but also with the claim construction standard applied in other post-grant procedures.
While the on-the-ground implications of the rule change will remain unclear until the rule change has been in effect for a longer period of time, the Federal Circuit has noted in the past that applying the broadest reasonable interpretation claim construction standard makes invalidation based on prior art easier. See, e.g., ePlus, Inc. v. Lawson Software, Inc., 790 F.3d 1307, 1314 (Fed. Cir. 2015) (noting that the "claim construction construct -- broadest reasonable interpretation --  makes invalidation based on prior art easier."). Based on this theory, and the Federal Circuit's directives described above, one possible on-the-ground effect of this potential disharmony in claim construction standards is that it would be easier to reject a new claim as anticipated by or obvious in light of prior art at the PTO in front of a patent examiner (because the examiner is applying a broader construction standard the sweeps in more prior art), than it would be to reject that same claim in light of prior art at the PTAB as submitted in a motion to amend (because the PTAB is applying a narrower construction standard that avoids certain prior art). This would mean, conversely, that it would be harder to invalidate a new claim submitted in a motion to amend at the PTAB (and therefore easier to obtain a new claim as a motion to amend applicant), than it would be for a patent examiner to reject that same claim if submitted in a patent application.
This potential effect may be entirely theoretical if, as pointed out by various practitioners, certain Federal Circuit panels, and select PTAB judges, there is really no difference in claim construction outcome regardless of which standard is applied. Examples of cases in which the standard applied did not make a difference to the outcome of claim construction include Black & Decker, Inc. v. Positec USA, Inc., 646 F. App'x 1019, 1024 (Fed. Cir. 2016) (noting that the PTAB erroneously applied the broadest reasonable interpretation standard to an expired patent, but holding that, "[d]espite the Board's use of an improper standard, applying the Phillips standard, we find that its ultimate construction of 'fixedly secured/securing' is nonetheless correct."); Straight Path IP Grp., Inc. v. Sipnet EU S.R.O., 806 F.3d 1356, 1360 (Fed. Cir. 2015) ("Straight Path ... asks us to determine the governing construction under the principles of Phillips ... , rather than the broadest-reasonable-interpretation standard. ... We need not explore the issues raised by that request, however, because we conclude that the Board adopted a claim construction that is erroneous even under the broadest-reasonable-interpretation standard."); In re CSB-System International, Inc., 832 F. 3d 1335, 1337-38 (Fed. Cir. 2016) ("We agree with CSB that the Board should have applied the Phillips standard of claim construction rather than the broadest reasonable interpretation standard used by the examiner ... We conclude, however, that the Board's claim construction was correct even under the Phillips standard."); and Cisco Systems, Inc. v. AIP Acquisition, LLC, IPR2014-00247, Paper 20 at 3 (July 10, 2014) ("We have determined, on the record before us, that the claim construction of the three phrases ... under the rule of construction similar to that applied by the district courts, ... are the same as the claim construction for those phrases under the rule of broadest reasonable interpretation"). However, even if this may be the case in certain circumstances, if it were universally true, then that would mean the PTO just went through a whole lot of effort to change the standard applied by the PTAB in America Invents Act proceedings for no practical purpose, which is unlikely. Indeed, the standard applied often does make a difference in outcome. See, e.g., PPC Broadband, Inc. v. Corning Optical Communications RF LLC, 815 F.3d 734, 741 (Fed. Cir. 2016) ("This case hinges on the claim construction standard applied -- a scenario likely to arise with frequency. And in this case, the claim construction standard is outcome determinative."). Moreover, by its own, most recent statistics based on AIA trial data through March 31, the PTAB has only allowed claim amendments (granting or granting-in-part) 10 percent of the time in response to motions to amend filed by patent owners. This provides further reason to believe that any potential practical effect of the recent rule change on the issuance of new claims at the PTAB would be minimal, if present at all.
If it turns out that the rule change does, in fact, create a more-than-theoretical effect on the issuance new claims at the PTAB, the significance of this potential effect may be heightened by PTO Director Andrei Iancu's recent statements on the rule change made in his speech at the American Intellectual Property Law Association's annual meeting on Oct. 25. During the meeting, Iancu emphasized that the current system "is not working as intended" and that the PTO is proposing a "robust new amendment process" to address the current sentiment among patent owners that motions to amend submitted during inter partes review proceedings are always unsuccessful. Iancu specifically noted that "[s]ome have suggested that parties have simply stopped even trying to amend the claims because they see the effort as largely futile," but "the AIA statute specifically provides for claim amendments in [inter partes reviews], so in order to fully implement the intent of the AIA, we must find a way to make this amendment process feasible and meaningful." Under the proposed new amendment rules, after a compressed 12-week schedule for motion practice, the PTAB would issue a preliminary ruling on the proposed amendments to which both parties could respond prior to receiving the PTAB's final ruling. Iancu explained that this procedure "should lead to more narrowly tailored and focused claim amendments, and potential earlier resolution of the issues." Accordingly, if these new procedures for motions to amend are put into effect and patent owners are incentivized to file more motions to amend, then we may see even more test grounds for new "substitute claims" to be granted at the PTAB using the narrower Phillips standard that may not have been granted by a patent examiner using the broadest reasonable standard.
The PTO's rule change just took effect on Nov. 13, so we will soon see whether this potential source of disharmony regarding claim construction standards applied to new claims will cause any on-the-ground effects on claim issuance at the PTAB.

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Staphylococcus aureus has been shown to invade and induce the death of various cell types. Here we investigate whether the cytotoxicity of intracellular S. aureus is a general feature or rather characteristic of individual S. aureus strains. The majority of 23 randomly collected clinical S. aureus isolates were killed inside keratinocytes and fibroblasts, indicating that the uptake of S. aureus represents an important mechanism of cell-autonomous host defense. However, seven independent S. aureus isolates survived intracellularly and induced significant cytotoxicity for their host cells. Subcloning analysis revealed that the ability or inability to kill host cells is a stable, apparently genetically determined trait of a given S. aureus isolate. We show that noncytotoxic strains but not cytotoxic strains colocalize with the lysosomal marker LAMP-1, suggesting that only cytotoxic strains escape degradation by the endolysosomal pathway. In a mouse septicemic model, cytotoxic S. aureus isolates produce significantly greater lethality (96%) compared to noncytotoxic strains (41%), which corresponds to 23-, 63-, and 30,000-fold increases of bacterial loads in the liver, spleen, and kidney, respectively. Finally, cytotoxic S. aureus strains produce clinically apparent arthritis in mice at a greater frequency than compared to noncytotoxic S. aureus strains. The results of our study unravel a previously unrecognized dichotomy of cytotoxic and noncytotoxic S. aureus isolates, which may play an important role in the dissemination of, and mortality induced by, S. aureus infection.
Staphylococcus aureus is one of the most important human pathogens, which causes both community-acquired and nosocomial infections (12). The diseases caused by this bacterium range from wound and soft tissue infections to endocarditis and septic shock. To defeat the innate and the adaptive immune system of the host, S. aureus employs both single-gene-encoded virulence factors such as alpha-toxin, coagulase, and protein A, as well as complex mechanisms such as adhesion or slime production. Although S. aureus is generally classified as an extracellular pathogen, recent data revealed its ability to infect various types of host cells: both professional phagocytes and nonphagocytes, including endothelial cells, fibroblasts, and others (1, 15, 25). This invasion is initiated by the adherence of S. aureus to the cellular surface, a process in which staphylococcal fibronectin-binding proteins play a prominent role (11, 14, 26, 30). Phagocytosed S. aureus can either induce apoptosis of the host cell (4) or survive for several days intracellularly in the cytoplasm, which is thought to be devoid of antistaphylococcal effector mechanisms (2, 28). Variants of S. aureus, termed small-colony variants (SCV) due to their colony morphology, have been reported to be especially able to persist intracellularly (35, 36). Although S. aureus has been shown to escape from the phagosome (4), neither the kinetics of escape nor the underlying mechanisms employed by S. aureus have yet been determined. Moreover, it is still unclear whether invasion and cytotoxicity are a common feature of clinical S. aureus isolates and whether these factors contribute to pathogenicity.
We show here that each of 23 randomly selected clinical S. aureus isolates infects nonphagocytic host cells. However, after uptake into the cell, the fate of both the bacteria and the host cells are shown to depend on the individual S. aureus strain. Specifically, a significant fraction of the clinical isolates killed more than 50% of the host cells within 24 h. We finally demonstrate that this in vitro cytotoxicity strongly correlates with the pathogenicity in mice, suggesting that the trait of cytotoxicity has to be considered as an important individual virulence factor of a given S. aureus strain.
Antibodies.Lysosomes were stained with anti-LAMP-1 monoclonal antibody (BD Pharmingen, San Diego, Calif.), followed by Cy3 (Amersham Biosciences)-coupled anti-rat immunoglobulin G secondary antibody (Dianova, Hamburg, Germany). Nonlabeled intracellular S. aureus were stained with anti-protein A antibody (Sigma, Taufkirchen, Germany) followed by Cy3-coupled anti-mouse immunoglobulin G secondary antibody (Dianova).
Bacteria.A total of 23 randomly selected S. aureus clinical isolates, as well as the ATCC 29213 reference strain, were used throughout the present study. All S. aureus isolates were typed by RAPD [random(ly) amplified polymorphic DNA] PCR to ascertain unrelatedness of origin. S. aureus grown overnight at 37°C in Luria-Bertani broth were diluted with fresh broth and cultured until mid-logarithmic phase of growth (optical density at 600 nm = 0.3). Bacteria were harvested, washed with PBSE (phosphate-buffered saline [PBS], 5 mM EDTA), and adjusted to 109 CFU/ml. The pair of S. aureus DU1090 strains stably expressing wild-type or mutant H35R amino acid substitution alpha-toxin was generously provided by S. Bhakdi (18).
RAPD PCR analysis.Genomic DNA was prepared by using DNeasy tissue kit (Qiagen, Hilden, Germany). PCR was performed by using Ready-To-Go RAPD PCR kit with Primer1 (GGTGCGGGGAA) according to the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany).
FITC staining of S. aureus.Bacterial pellets of 1010 CFU were resuspended in carbonate buffer (pH 9.0) containing 100 μg of fluorescein isothiocyanate (FITC) isomer I (Sigma, Taufkirchen, Germany)/ml for 1 h at room temperature. Staphylococci were extensively washed with PBSE, adjusted with 10% glycerol to 109 CFU/ml, and kept at −70°C until use. No significant loss of viability or fluorescence intensity during the freeze-thaw procedure was observed.
Eukaryotic cell culture.Mouse keratinocyte PAM212 and fibroblast mKSA cell lines were cultured, respectively, in RPMI 1640 and Dulbecco modified Eagle medium (both from Biochrom, Berlin, Germany). Both media were supplemented with 10% heat-inactivated fetal calf serum (Biochrom), 100 U of penicillin G/ml, and 100 μg of streptomycin sulfate (Biochrom)/ml. At 18 h prior to infection, 5 × 105 cells were seeded in six-well plates (total volume = 3 ml) (Nunc, Wiesbaden, Germany). Viability was monitored by using trypan blue (Biochrom) exclusion (7).
Invasion assay.Prior to infection, cells were washed with growth medium without antibiotics and kept for 1 h at 37°C. A total of 106 to 108 FITC-stained staphylococci were added per well. After a 1 h of incubation, cells were detached from the culture plate and washed with growth medium. Extracellular staphylococci were killed by incubation of cells with 100 μg of lysostaphin (Sigma)/ml for 7 min at 37°C. Cells were washed with growth medium supplemented with antibiotics and reseeded in a new six-well plate (total volume = 3 ml). After 24 h of incubation, cells were harvested and analyzed by flow cytometry and microscopy. Host cell viability was measured by using trypan blue exclusion (7). For fluorescence microscopy, cells were grown on coverslips. After the invasion procedure, cells were rinsed with PBS and fixed for 15 min with 4% paraformaldehyde. Cells were permeabilized with 0.1% saponin prior to staining with antibodies specific for organelles or intracellular bacteria. Specimens were mounted on microscopy slides in 10% glycerol supplemented with 100 mg of DABCO (diazabicyclo[2.2.2]octane; Sigma)/ml. Images were acquired by using an Axioscop 2 microscope (Zeiss, Oberkochen, Germany) equipped with an Axiocam charge-coupled device camera and then analyzed by using Axiovision software (Zeiss).
Flow cytometry.The supernatants of the infected cultures were removed to preserve detached cells. Subsequently, the adherent cells were harvested by standard trypsin treatment and combined with cells from the supernatants. Cell samples were washed once with PBS and submitted to analysis by flow cytometry. Noninfected cells served as negative control to set the cutoff marker for the discrimination of FITC-negative and positive cells.
CFU determination.Infected cells were lysed in PBSE containing 0.05% Triton X-100. Lysates were sonicated for 5 min at 4°C. This procedure was found to be most effective to resolve the bacterial clumps. Then, lysates were diluted with PBS and plated on Mueller-Hinton agar by using a spiral plater (EDDY-Jet; IUL Instruments, Königswinter, Germany). Colony counting and CFU determination were performed according to the instructions of the manufacturer.
Mouse infection model.C57BL/6N Crl BR mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and kept under barrier conditions. Mice of 6 to 10 weeks age were infected by intravenous (i.v.) injection of graded doses of S. aureus in 0.3 ml of PBS. Mice were monitored daily for clinical signs of S. aureus infection (ruffled fur, reduced activity, hunched posture) and mortality. Arthritis induced by S. aureus infection was defined as the impairment of the mobility in major joints leading to alterations of the gait (8). In order to determine the bacterial load in various organs, mice were sacrificed 24 h after infection. Livers, spleens, and kidneys were removed under aseptic conditions and then homogenized in 0.05% Triton X-100, sonicated for 5 min at 4°C, and plated on blood agar by using a spiral plater.
Statistical analysis.Experimental data were assessed with a two-tailed unpaired Student t test for comparison between means. Arthritis frequencies and survival data were compared by using the Mann-Whitney U test. P values of <0.01 were considered to be statistically significant.
Invasiveness and cytotoxicity of S. aureus.Up to the present, the ability of S. aureus to invade and induce the death of eukaryotic cells has been investigated by employing select host cell types and specific laboratory strains S. aureus such as NCTC 8325-4. To investigate whether invasion and cytotoxicity are general phenomena of S. aureus, 23 randomly selected clinical isolates were tested for their ability to infect either keratinocytes (PAM212) or fibroblasts (mKSA). RAPD PCR analysis revealed unrelatedness of all S. aureus isolates investigated (data not shown). To monitor the invasiveness of S. aureus, all 23 clinical isolates were FITC labeled and incubated with either PAM212 or mKSA cells at a multiplicity of infection (MOI) of 200. After 1 h, extracellular bacteria were destroyed by the addition of lysostaphin. The outgrowth of potentially surviving extracellular S. aureus was inhibited by penicillin. As shown in Fig. 1A, intracellular S. aureus were readily detected in PAM212 and mKSA cells by flow cytometry. To rule out that invasion was facilitated by FITC, nonlabeled S. aureus were incubated with PAM212 cells and stained with anti-protein A antibody. As shown in Fig. 1B, S. aureus was detected inside host cells by indirect immunofluorescence, indicating that the invasion of S. aureus occurs independently of FITC.
Each of the tested S. aureus clinical isolates proved able to invade both cell types with similar efficiency (Fig. 2A). However, only a fraction of these clinical isolates (7 of 23 [30%]) produced significant (>50% of host cells killed; P < 0.01) cytotoxicity 24 h after infection (Fig. 2B). It should be noted that invasion occurs rapidly upon incubation of the host cells with bacteria. In contrast, the cytotoxicity for host cells manifests more slowly to become visible only >4 h after infection (data not shown). The well-characterized reference strain ATCC 29213 belongs to the cytotoxic fraction of S. aureus. It is important to note that the cytotoxic potential of a given S. aureus strain appears to be a genetically determined trait. Subclones of any of these strains exerted the same degree of host cell cytotoxicity compared to the parental strain (Fig. 2C). The dichotomy of cytotoxic versus noncytotoxic strains of S. aureus also becomes apparent in dose-response studies. Although host cell killing by cytotoxic strains started at an MOI of 20, no signs of cytotoxicity were detected with noncytotoxic strains at MOIs of <500 (Fig. 2D). To prove that cell death was not induced during the 1-h incubation-invasion phase, we infected mKSA cells with heat-killed cytotoxic S. aureus. As shown in Fig. 2E, heat-killed bacteria were unable to induce cytotoxicity. Additionally, infected cells were treated with the plasma membrane-permeable antibiotic rifampin. As shown in Fig. 2E, rifampin added after the infection phase rescued the host cells from S. aureus-induced cell death. Thus, induction of host cell death requires death signals from metabolically active, intracellular S. aureus.
Intracellular fate of S. aureus.In infected host cells, only FITC-labeled cytotoxic S. aureus strains could be detected by flow cytometry after 24 h (Fig. 3A). In contrast, the fluorescence of cells infected with noncytotoxic S. aureus strains or with heat-killed cytotoxic S. aureus isolates rapidly declined to become undetectable within 24 h (Fig. 3B and C). Indeed, the recovery of viable bacteria rapidly declined in the case of noncytotoxic S. aureus without killing of the host cells (Fig. 3D). By 72 h postinfection, only minute numbers of CFU of noncytotoxic S. aureus could be detected, confirming the results obtained by flow cytometry. These findings indicate a different rate of elimination and suggested a different intracellular fate of cytotoxic versus noncytotoxic S. aureus strains.
Host cells kill intracellular bacteria by means of phagolysosomal fusion and subsequent degradation by oxygen radicals, NO, and proteases. Obligate and facultative intracellular bacteria, however, have developed effective mechanisms to avoid killing by host cells that include evasion of the phagosome and inhibition of phagolysosomal fusion. We thus addressed the question of whether cytotoxic and noncytotoxic S. aureus strains localize to the same or different subcellular compartments. As shown in Fig. 4A, after 3 h of infection noncytotoxic S. aureus colocalizes with the lysosomal marker antigen LAMP-1, suggesting that noncytotoxic S. aureus enters the lysosomal degradation pathway. Indeed, by 24 h the signal of FITC-labeled S. aureus remained associated with LAMP-1 and yet was significantly decreased, which may be explained by either quenching of FITC fluorescence at acidic pH, degradation of bacteria, or both. In contrast, cytotoxic S. aureus strains do not colocalize with LAMP-1 (Fig. 4B), that is, do not enter the lysosomal compartment. Like noncytotoxic S. aureus, heat-killed cytotoxic S. aureus also colocalized with LAMP-1 (Fig. 4C), indicating that escape from the lysosomal pathway and killing of host cells is an achievement of viable, metabolically active staphylococci rather than due to a preformed cytotoxic component of S. aureus. Specifically, no differences in intracellular cytotoxicity were observed with a pair of congenic wild-type alpha-toxin-producing and H35R alpha-toxin mutant S. aureus strains (data not shown), indicating that alpha-toxin is not responsible for the killing of host cells by intracellular S. aureus.
Enhanced virulence of cytotoxic S. aureus.In order to address the question of whether the distinction between cytotoxic and noncytotoxic S. aureus is a mere in vitro phenomenon or instead relates to in vivo virulence, mice were challenged i.v. with five representative cytotoxic or seven noncytotoxic S. aureus strains. Injection of 108 CFU of either cytotoxic or noncytotoxic S. aureus resulted within 24 h in the death of 21 of 23 mice and 4 of 27 mice, respectively (Fig. 5A and Table 1). The bacterial loads in the liver, spleen, and kidney of mice challenged with cytotoxic S. aureus were approximately 36-, 26-, and 30,000-fold greater (P < 0.01), respectively, than those observed with noncytotoxic S. aureus (Fig. 5B).
The enhanced virulence of cytotoxic S. aureus became also apparent when mice were infected with sublethal doses of either five cytotoxic or seven noncytotoxic S. aureus strains. As shown in Table 1, infection of mice with 106 CFU of cytotoxic S. aureus resulted in clinically apparent arthritis in the limbs of 73% of the animals, whereas the same infectious dose of noncytotoxic S. aureus produced arthritis in only 19% of the mice (P < 0.01). Together, these results indicate that the ability of S. aureus to kill host cells corresponds well with their replication, systemic dissemination, and organ infiltration in vivo.
S. aureus is one of the most prevalent bacterial pathogens isolated from patients with bloodstream infections. An important feature of S. aureus bacteremia is the frequency with which it penetrates from the bloodstream into other tissues such as bones and joints, the lungs, and the central nervous system (21). The rate of metastatic infection following S. aureus bacteremia varies from 1 to 53% depending on the authors and study with a median rate of 23.3% (16). The data of our present study show that 7 of 23 clinical S. aureus isolates after infection of keratinocytes and fibroblasts are able to escape phagolysosomal degradation, survive intracellularly, and produce significant cytotoxicity. The respective ability or inability to survive in and kill host cells turned out to be a stable trait of a given clinical S. aureus isolate. This suggests that the probability of metastatic infection in S. aureus bacteremia might be determined, at least in part, by the cytotoxicity of S. aureus. In fact, we observed significantly greater lethality, organ infiltration, and arthritis in mice challenged with cytotoxic S. aureus strains. We therefore propose that the ability to survive in and kill host cells may be a virulence factor that determines the propensity of S. aureus to negotiate cellular barriers and to penetrate from the bloodstream into tissues.
Using select pathogenic and nonpathogenic laboratory strains of S. aureus, it has been shown previously by many investigators that S. aureus can invade, survive in, and kill nonprofessional phagocytes such as endothelial cells, fibroblasts, keratinocytes, osteoblasts, and macrophages (4, 19, 23, 24, 33). This invasion is initiated by the adherence of S. aureus to the cellular surface, a process in which staphylococcal fibronectin-binding proteins play a prominent role (1, 11, 14, 15, 25, 26, 30). The adherent bacteria induce polymerization of cellular actin, leading to the engulfment by the plasma membrane, internalization via the “zipper mechanism” (13), and finally their transport within phagosomes into the cell (22). The uptake of S. aureus, for example, by endothelial cells, leads to the expression of adhesion molecules (5, 6, 10) and the secretion of proinflammatory cytokines (37). Phagocytosis of extracellular bacteria by professional phagocytes and internalization by nonprofessional phagocytes is generally viewed as a cell-autonomous defense mechanism. Several important human pathogens have developed effective antiphagocytic mechanisms, including encapsulation. The antiphagocytic activity of capsular polysaccharides and increased virulence of encapsulated strains were shown for Haemophilus influenzae, Pseudomonas aeruginosa, and Streptococcus pyogenes (9, 29, 31). Particularly, group A streptococci can be internalized by epithelial cells and seem to be trapped without producing any cytopathic effects (9). With regard to S. aureus, 90% of S. aureus strains express a capsule that is antiphagocytic (32), and capsule expression has been reported to negatively correlate with adherence to endothelial cells (27). However, all clinical isolates investigated are obviously able to invade nonprofessional phagocytes in equal quantities and with similar kinetics, indicating that possible differences in encapsulation do not impact on invasiveness.
In our study, we provide evidence that S. aureus strains can be distinguished by their survival abilities after internalization. For the majority of clinical S. aureus isolates (>70%), phagocytosis and subsequent lysosomal degradation seems to be a powerful means for host cells to eliminate these bacteria. This holds also true for SCV of S. aureus with reduced energy metabolism that have been reported to be especially adapted to survive intracellularly (35, 36). We have analyzed three clinical isolates of SCV. Although any of these SCV readily infected host cells, they were eliminated within 48 h and did not produce cytotoxicity (unpublished observations). These data indicate an obviously mutually exclusive relationship between S. aureus and host cells: either S. aureus is killed by the host cells and the host cell survives, or S. aureus remains viable and the host cells are killed.
By lysing their host cells, intracellular S. aureus once again become extracellular and can invade the adjacent tissue. Our mouse models demonstrate that, compared to noncytotoxic strains, cytotoxic S. aureus produce greater lethality; dramatically increased bacterial loads in liver, spleen, as well as kidney; and induced arthritis at greater frequency. In order to spread into various organs, circulating S. aureus organisms have to pass the endothelial barrier. We therefore conclude that cytotoxic S. aureus may disrupt the integrity of the endothelial barrier, facilitating bacterial metastasis and seeding. With regard to S. aureus-induced arthritis formation, Tarkowsky and coworkers recently implied that peptidoglycan was a driving force (20). According to the results of our study this might be secondary to cytolysis of cellular barriers and bacterial invasion of joints.
Up to the present, the molecular mechanisms of S. aureus-mediated killing of host cells remained controversial. Staphylococcal alpha-toxin has been implicated as the main factor in lysing endothelial cells (23). This soluble, pore-forming protein of S. aureus reported to induce apoptosis via the caspase death pathway (3). Bantel et al. noted that alpha-toxin present in the supernatant of S. aureus cultures induced cytotoxicity that does not require bacterial internalization (3). This finding is in striking contrast to previous reports by many investigators who proposed that S. aureus-induced apoptosis requires internalization of bacteria (4, 19, 23, 24, 33). These contradictory findings may reflect a cell-type-specific phenomenon, because compared to other cell types lymphocytes have been shown to be especially sensible to killing by alpha-toxin (17, 34). In addition, the observations by Bantel et al. may be secondary to unphysiological alpha-toxin concentrations used for cell death studies. In their experimental approach, supernatants of 14-h S. aureus cultures were used, which are stationary, well above quorum sensing, and which, albeit filtered, probably contain significant amounts of toxic molecules derived from dead bacteria. Whether circulating S. aureus can accumulate to such large numbers and produce comparable amounts of alpha-toxin is questionable. In fact, we did not observe a correlation between the expression levels of alpha-toxin and the cytotoxic activity of the various clinical S. aureus isolates. Specifically, a laboratory strain selected for high production of alpha-toxin did not cause host cell death, indicating that alpha-toxin production inside host cells does not suffice to initiate apoptotic cell death. The fact that rifampin rescued host cells previously infected with cytotoxic S. aureus provides strong evidence that S. aureus can kill their host cells from inside, that is, after internalization.
We thank Herdis Sommer for excellent technical assistance. We thank S. Bhakdi for providing isogenic alpha-toxin-producing and mutant S. aureus strains and for helpful discussion.
This work was partially supported by the DFG (SFB 589) and Maria Pesch Stiftung, Cologne, Germany.
Returned for modification 22 October 2002.
Almeida, R. A., K. R. Matthews, E. Cifrian, A. J. Guidry, and S. P. Oliver. 1996. Staphylococcus aureus invasion of bovine mammary epithelial cells. J. Dairy Sci. 79:1021-1026.
Balwit, J. M., P. van Langevelde, J. M. Vann, and R. A. Proctor. 1994. Gentamicin-resistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells. J. Infect. Dis. 170:1033-1037.
Bantel, H., B. Sinha, W. Domschke, G. Peters, K. Schulze-Osthoff, and R. U. Janicke. 2001. α-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling. J. Cell Biol. 155:637-648.
Beekhuizen, H., J. S. van de Gevel, B. Olsson, I. J. van Benten, and R. van Furth. 1997. Infection of human vascular endothelial cells with Staphylococcus aureus induces hyperadhesiveness for human monocytes and granulocytes. J. Immunol. 158:774-782.
Bengualid, V., V. B. Hatcher, B. Diamond, E. A. Blumberg, and F. D. Lowy. 1990. Staphylococcus aureus infection of human endothelial cells potentiates Fc receptor expression. J. Immunol. 145:4279-4283.
Bonifacio, J. S. (ed.). 2000. Current protocols in cell biology. John Wiley & Sons, Inc., New York, N.Y.
Bremell, T., S. Lange, A. Yacoub, C. Ryden, and A. Tarkowski. 1991. Experimental Staphylococcus aureus arthritis in mice. Infect. Immun. 59:2615-2623.
Cywes, C., and M. R. Wessels. 2001. Group A streptococcus tissue invasion by CD44-mediated cell signalling. Nature 414:648-652.
Drake, T. A., and M. Pang. 1988. Staphylococcus aureus induces tissue factor expression in cultured human cardiac valve endothelium. J. Infect. Dis. 157:749-756.
Dziewanowska, K., J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach. 1999. Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infect. Immun. 67:4673-4678.
Emori, T. G., and R. P. Gaynes. 1993. An overview of nosocomial infections, including the role of the microbiology laboratory. Clin. Microbiol. Rev. 6:428-442.
Fowler, T., E. R. Wann, D. Joh, S. Johansson, T. J. Foster, and M. Hook. 2000. Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell β1 integrins. Eur. J. Cell Biol. 79:672-679.
Ing, M. B., L. M. Baddour, and A. S. Bayer. 1997. Bacteremia and infective endocarditis: pathogenesis, diagnosis, and complications, p. 331-354. In K. B. Crossley and G. L. Archer (ed.), The staphylococci in human disease. Churchill Livingstone, New York, N.Y.
Jonas, D., I. Walev, T. Berger, M. Liebetrau, M. Palmer, and S. Bhakdi. 1994. Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation. Infect. Immun. 62:1304-1312.
Jursch, R., A. Hildebrand, G. Hobom, J. Tranum-Jensen, R. Ward, M. Kehoe, and S. Bhakdi. 1994. Histidine residues near the N terminus of staphylococcal alpha-toxin as reporters of regions that are critical for oligomerization and pore formation. Infect. Immun. 62:2249-2256.
Liu, Z. Q., G. M. Deng, S. Foster, and A. Tarkowski. 2001. Staphylococcal peptidoglycans induce arthritis. Arthritis Res. 3:375-380.
Lowy, F. D., J. Fant, L. L. Higgins, S. K. Ogawa, and V. B. Hatcher. 1988. Staphylococcus aureus-human endothelial cell interactions. J. Ultrastruct. Mol. Struct. Res. 98:137-146.
Nuzzo, I., M. R. Sanges, A. Folgore, and C. R. Carratelli. 2000. Apoptosis of human keratinocytes after bacterial invasion. FEMS Immunol. Med. Microbiol. 27:235-240.
Ogawa, S. K., E. R. Yurberg, V. B. Hatcher, M. A. Levitt, and F. D. Lowy. 1985. Bacterial adherence to human endothelial cells in vitro. Infect. Immun. 50:218-224.
Peacock, S. J., T. J. Foster, B. J. Cameron, and A. R. Berendt. 1999. Bacterial fibronectin-binding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus aureus to resting human endothelial cells. Microbiology 145:3477-3486.
Pohlmann-Dietze, P., M. Ulrich, K. B. Kiser, G. Doring, J. C. Lee, J. M. Fournier, K. Botzenhart, and C. Wolz. 2000. Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase. Infect. Immun. 68:4865-4871.
Proctor, R. A., J. M. Balwit, and O. Vesga. 1994. Variant subpopulations of Staphylococcus aureus as cause of persistent and recurrent infections. Infect. Agents Dis. 3:302-312.
Ruhen, R. W., P. G. Holt, and J. M. Papadimitriou. 1980. Antiphagocytic effect of Pseudomonas aeruginosa exopolysaccharide. J. Clin. Pathol. 33:1221-1222.
Sinha, B., P. P. Francois, O. Nusse, M. Foti, O. M. Hartford, P. Vaudaux, T. J. Foster, D. P. Lew, M. Herrmann, and K. H. Krause. 1999. Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin α5β1. Cell Microbiol. 1:101-117.
St Geme, J. W., III, and S. Falkow. 1992. Capsule loss by Haemophilus influenzae type b results in enhanced adherence to and entry into human cells. J. Infect. Dis. 165(Suppl. 1):S117-S118.
Thakker, M., J. S. Park, V. Carey, and J. C. Lee. 1998. Staphylococcus aureus serotype 5 capsular polysaccharide is antiphagocytic and enhances bacterial virulence in a murine bacteremia model. Infect. Immun. 66:5183-5189.
Tucker, K. A., S. S. Reilly, C. S. Leslie, and M. C. Hudson. 2000. Intracellular Staphylococcus aureus induces apoptosis in mouse osteoblasts. FEMS Microbiol. Lett. 186:151-156.
Valeva, A., I. Walev, M. Pinkernell, B. Walker, H. Bayley, M. Palmer, and S. Bhakdi. 1997. Transmembrane beta-barrel of staphylococcal alpha-toxin forms in sensitive but not in resistant cells. Proc. Natl. Acad. Sci. USA 94:11607-11611.
Vesga, O., M. C. Groeschel, M. F. Otten, D. W. Brar, J. M. Vann, and R. A. Proctor. 1996. Staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu. J. Infect. Dis. 173:739-742.
von Eiff, C., K. Becker, D. Metze, G. Lubritz, J. Hockmann, T. Schwarz, and G. Peters. 2001. Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier's disease. Clin. Infect. Dis. 32:1643-1647.
Yao, L., V. Bengualid, F. D. Lowy, J. J. Gibbons, V. B. Hatcher, and J. W. Berman. 1995. Internalization of Staphylococcus aureus by endothelial cells induces cytokine gene expression. Infect. Immun. 63:1835-1839.


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Bust of Don Guillermo V Flores at Villa Flores in Piddig.
Don Guillermo V. Flores was born February 9, 1881 in Piddig Ilocos Norte, the town of the Basi Rebellion. He was married to Doña Adelaida Duque at an early age. They were blessed with ten children. He has shown a keen interest to learn and study the Grammatica Castellana (Spanish grammar), Geography and Arithmetica in the private school of his father, Don Cornelioi Flores, a maestro normalista and a native of Vintar, Ilocos Norte during the Spanish regime. He went to farther his study when the American Government took over in the Philippines.
Don Guillermo V. Flores was born with a golden pen in his hand, with a spirit that is noble, a spirit that is artistic and a spirit that is revolutionary—a devoted Patriarch, Consumate, Politician, accomplished Artist, Poet, Writer, Educator and Composer rolled into one.
In 1907, he became a Maestro (teacher) in the school of the American Military Government when there wasn’t a civil government established in the Philippines. Soon he became the Escritibiente Municipal from 1899 to the year 1903.
He was designated as Enumerador (Enumerator) of the first census in the Philippines. He became a Notario Publico (Notary Public) and was elected Vise Presidente Municipal (Vice Mayor) in 1913. He became the Presidente Municipal (Municipal Mayor) from 1916 to 1920. From 1935 to 1939, he was elected as Concejal Municipal (Municipal Councilman).
During the Filipino-American war, Don Guillermo V. Flores served in the Segunda Compania Batallion Ilocos Norte and was subsequently promoted to the rank of Kapitan (Captain) in the Association de los Veteranos de la Revolotion Filipina headed by General Emilio Aguinaldo.
During his administration from 1916 to 1920 the Municipal Council was constituted with Don Guillermo V. Flores as Municipal Presidente. Significant developments took place in Piddig. Under the leadership of Don Guillermo V. Flores, he established the Escuela Gabaldon (now the Piddig Central Elementary School) and the Presidencia Municipal which was later on improved and named Capitol Hill by the former Congressman of the First District, Governor of Ilocos Norte, Secretary of Public Works and Highways and Parliamentarian, the late Antonio V. Raquiza.
In the field of literary arts, Don Guillermo V. Flores produced some immortal Ilocano classics most notably, the song, Simngay ka Piddig. The song which evokes a strong sense of nostalgia among all Piddiguenos wherever they may be in the world and whenever they come together to relive past memories, to renew old ties, or just simply feel homesick. This song has now become the binding tie that unites us Piddiguenos, and the obra maestro that defines what Piddig is and its people.
He is also well remembered for some of the best Ilocano love songs and poems such as Canito ni Ayat, Sika Gayam, Guita ni Ayat, Diak kad Basol. Sueltos (Zarzuelas) some of which he worked closesly with Don Pascual Agcaoili another famous Zarzuelita of Piddig whose work was staged at the Cultural Center in the Philippines.
Don Guillermo V. Flores died at the age of 96 on February 18, 1977.

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§ 4.2 Payment of minimum wage specified in section 6(a)(1) of the Fair Labor Standards Act of 1938 under all service contracts.
§ 4.4 Obtaining a wage determination.
§ 4.5 Contract specification of determined minimum wages and fringe benefits.
§ 4.6 Labor standards clauses for Federal service contracts exceeding $2,500.
§ 4.10 Substantial variance proceedings under section 4(c) of the Act.
§ 4.11 Arm's length proceedings.
§ 4.12 Substantial interest proceedings.
§ 4.1a Definitions and use of terms.
§ 4.1b Payment of minimum compensation based on collectively bargained wage rates and fringe benefits applicable to employment under predecessor contract.
§ 4.50 Types of wage and fringe benefit determinations.
§ 4.51 Prevailing in the locality determinations.
§ 4.52 Fringe benefit determinations.
§ 4.53 Collective bargaining agreement (successorship) determinations.
§ 4.54 Locality basis of wage and fringe benefit determinations.
§ 4.55 Issuance and revision of wage determinations.
§ 4.56 Review and reconsideration of wage determinations.
§ 4.101 Official rulings and interpretations in this subpart.
§ 4.102 Administration of the Act.
§ 4.104 What the Act provides, generally.
§ 4.105 The Act as amended.
§ 4.108 District of Columbia contracts.
§ 4.110 What contracts are covered.
§ 4.115 Exemptions and exceptions, generally.
§ 4.116 Contracts for construction activity.
§ 4.117 Work subject to requirements of Walsh-Healey Act.
§ 4.118 Contracts for carriage subject to published tariff rates.
§ 4.119 Contracts for services of communications companies.
§ 4.120 Contracts for public utility services.
§ 4.121 Contracts for individual services.
§ 4.122 Contracts for operation of postal contract stations.
§ 4.123 Administrative limitations, variances, tolerances, and exemptions.
§ 4.130 Types of covered service contracts illustrated.
§ 4.131 Furnishing services involving more than use of labor.
§ 4.132 Services and other items to be furnished under a single contract.
§ 4.133 Beneficiary of contract services.
§ 4.134 Contracts outside the Act's coverage.
§ 4.140 Significance of contract amount.
§ 4.141 General criteria for measuring amount.
§ 4.142 Contracts in an indefinite amount.
§ 4.143 Effects of changes or extensions of contracts, generally.
§ 4.144 Contract modifications affecting amount.
§ 4.145 Extended term contracts.
§ 4.146 Contract obligations after award, generally.
§ 4.150 Employee coverage, generally.
§ 4.151 Employees covered by provisions of section 2(a).
§ 4.152 Employees subject to prevailing compensation provisions of sections 2(a)(1) and (2) and 4(c).
§ 4.153 Inapplicability of prevailing compensation provisions to some employees.
§ 4.154 Employees covered by sections 2(a)(3) and (4).
§ 4.155 Employee coverage does not depend on form of employment contract.
§ 4.156 Employees in bona fide executive, administrative, or professional capacity.
§ 4.159 General minimum wage.
§ 4.160 Effect of section 6(e) of the Fair Labor Standards Act.
§ 4.161 Minimum monetary wages under contracts exceeding $2,500.
§ 4.162 Fringe benefits under contracts exceeding $2,500.
§ 4.163 Section 4(c) of the Act.
§ 4.165 Wage payments and fringe benefits—in general.
§ 4.166 Wage payments—unit of payment.
§ 4.167 Wage payments—medium of payment.
§ 4.168 Wage payments—deductions from wages paid.
§ 4.169 Wage payments—work subject to different rates.
§ 4.170 Furnishing fringe benefits or equivalents.
§ 4.171 “Bona fide” fringe benefits.
§ 4.172 Meeting requirements for particular fringe benefits—in general.
§ 4.173 Meeting requirements for vacation fringe benefits.
§ 4.174 Meeting requirements for holiday fringe benefits.
§ 4.175 Meeting requirements for health, welfare, and/or pension benefits.
§ 4.176 Payment of fringe benefits to temporary and part-time employees.
§ 4.177 Discharging fringe benefit obligations by equivalent means.
§ 4.178 Computation of hours worked.
§ 4.179 Identification of contract work.
§ 4.180 Overtime pay—in general.
§ 4.181 Overtime pay provisions of other Acts.
§ 4.182 Overtime pay of service employees entitled to fringe benefits.
§ 4.183 Employees must be notified of compensation required.
§ 4.184 Posting of notice.
§ 4.187 Recovery of underpayments.
§ 4.188 Ineligibility for further contracts when violations occur.
§ 4.189 Administrative proceedings relating to enforcement of labor standards.
§ 4.191 Complaints and compliance assistance.
41 U.S.C. 351 et seq.; 41 U.S.C. 38 and 39; 5 U.S.C. 301; Pub. L. 104–188, §2105(b); Pub. L. 110–28, 121 Stat. 112; Secretary's Order 9–2009, 74 FR 58836 (Nov. 13, 2009).
48 FR 49762, Oct. 27, 1983, unless otherwise noted.
, Nomenclature changes to part 4 appear at 61 FR 19984, May 3, 1996.


1. Where a claim of violation of federal right, based on the alleged action of the trial court in entering judgment without notice and an opportunity to be heard, was raised for the first time upon petition for rehearing (denied without opinion) in the state supreme court, although the same ground of objection had been raised throughout the proceedings, but solely as a question of state law, a writ of certiorari to review the judgment will be dismissed for failure to make seasonably the federal claim. P. 287 U. S. 162.
2. Where, upon the claim of a party that judgment was entered against him without jurisdiction in a state court, there is an adequate state remedy available, which he invokes and pursues to final judgment, the remedy by suit in the federal court is barred. P. 287 U. S. 164.
3. Where a judgment is attacked as having been entered without jurisdiction, an appeal from an order on motion to vacate, made on a general appearance, was effective to confer jurisdiction upon the state supreme court to determine whether the trial court had jurisdiction. P. 287 U. S. 165.
4. The full faith and credit clause applies to judicial proceedings of a state court drawn in question in an independent proceeding in the federal courts. P. 287 U. S. 166.
5. Principles of res judicata apply to questions of jurisdiction as well as to other issues. P. 287 U. S. 166.
6. Principles of res judicata may apply though proceeding was begun by motion. P. 287 U. S. 166.
7. Decision of state supreme court wherein question of jurisdiction of trial court to enter judgment was adjudicated on appeal in a proceeding begun by motion to set the judgment aside held bar to proceeding in federal court to enjoin enforcement of judgment for want of jurisdiction. Pp. 287 U. S. 166-167.
8. While due process requires that one against whom liability on a supersedeas bond is sought to be enforced shall have opportunity to present every available defense, this need not be before entry of judgment, and a state may constitutionally provide for such a hearing by an appeal after entry of judgment. P. 287 U. S. 168.
9. Where opportunity to raise issue of lack of notice in state courts was lost through failure seasonably to pursue appropriate state remedy, same issue cannot be utilized as basis for relief in federal court. P. 287 U. S. 169.
50 Idaho 606, certiorari dismissed.
Writs of certiorari, 286 U.S. 536537, to review judgments of the Supreme Court of Idaho and the Circuit Court of Appeals, involving the validity of a judgment against the surety company on a supersedeas bond. For opinion of federal district court, affirmed here, see 51 F.2d 596.
court for $22,357.21 and interest, on a supersedeas bond. No. 3, which is here on certiorari to the Supreme Court of Idaho, brings the record of the cause in which that judgment was entered. 286 U.S. 536. No. 21 is here on certiorari to the United states Circuit Court of Appeals for the Ninth Circuit, which reversed the decree of the Federal Court for Idaho denying the surety company's application to enjoin the enforcement of the judgment and dismissing the bill. 286 U.S. 537. In each case, it is claimed that the judgment is void under the due process clause of the Fourteenth Amendment.
"if the said appellant does not make such payment within thirty days from the filing of the remittitur from the Supreme Court in the court from which the appeal is taken, judgment may be entered on motion of the respondents in their favor against the undersigned surety. "
The Supreme Court affirmed the judgment as to Anderson, and reversed it as to the Singer Company. Baldwin v. Singer Sewing Machine Co. and Anderson, 49 Idaho 231. Upon the filing of the remittitur, the appropriate new judgment against Anderson was entered in the trial court. That judgment having remained unpaid more than thirty days, the Baldwins, without giving notice to either of the original defendants or to the surety company, moved the trial court to enter judgment against the latter. On June 23, 1930, judgment was so entered against the surety company in the sum of $22,357.21 and interest, with a provision "that the plaintiffs have execution therefor."
did not stay the judgment as against Anderson, but solely as against the Singer Company; that, hence, the surety company had not consented to the entry of a judgment against it upon Anderson's failure to pay, and that, since the judgment against it was entered without giving it notice and the opportunity of a hearing on the construction and effect of the bond, the judgment is void under the due process clause of the Fourteenth Amendment.
"Did the surety company, in its undertaking, become a party liable for every part of the judgment appealed from which might be affirmed by the Supreme Court, or did it stipulate only as to such judgment or part thereof as might be affirmed against the Singer Sewing Machine Company?"
that the trial court thus had the power and duty to construe the bond; that, "whether it decided right or wrong, its decision was a judgment which could be reviewed for error, if there was error, only by" the Supreme Court on appeal, and that the alleged error could not be raised on motion to vacate. 50 Idaho 609, 614-616.
without giving notice and an opportunity to be heard. The same ground of objection had been raised throughout the proceedings, but solely as a matter of state law. There had been ample opportunity earlier to present the objection as one arising under the Fourteenth Amendment. Compare Corkran Oil Co. v. Arnaudet, 199 U. S. 182, 199 U. S. 193; Godchaux Co. v. Estopinal, 251 U. S. 179, 251 U. S. 181; Live Oak Water Users' Assn. v. Railroad Comm'n, 269 U. S. 354, 269 U. S. 357. This is not a case where, as in Saunders v. Shaw, 244 U. S. 317, 244 U. S. 320, the federal claim arose from the unanticipated disposition of the case at the close of the proceedings in the state supreme court. Compare Ohio ex rel. Bryant v. Akron Metropolitan Park Dist., 281 U. S. 74, 281 U. S. 79. Nor is the federal claim based, as in Brinkerhoff-Faris Trust & Savings Co. v. Hill, 281 U. S. 673, 281 U. S. 678, upon the unanticipated act of the state supreme court in giving to a statute a new construction which threatened rights under the Constitution. Compare Missouri ex rel. Missouri Insurance Co. v. Gehner, 281 U. S. 313, 281 U. S. 320.
Second. In No. 21, the Circuit Court of Appeals should have affirmed the decree of the Federal Court for Idaho which denied the surety company's application for an interlocutory injunction and dismissed the bill. For the federal remedy was barred by the proceedings taken in the state court which ripened into a final judgment constituting res judicata.
Fe Ry. Co. v. Wells, 265 U. S. 101; Firestone Tire & Rubber Co. v. Marlboro Cotton Mills, 282 F. 811, 814. But an adequate state remedy was available, and, having invoked that and pursued it to final judgment, the surety company cannot escape the effect of the adjudication there. Compare Mitchell v. First National Bank, 180 U. S. 471, 180 U. S. 480-481; Lion Bonding Co. v. Karatz, 262 U. S. 77, 262 U. S. 90.
"construe the legal effect of the undertaking in question further than to examine it in aid of determining the sole question of the court's jurisdiction to hear and determine the motion for judgment thereon."
asserts, reach its decision by merely assuming the point in issue, or by deeming itself concluded by the fact that the trial court took jurisdiction. That it did not so reach its decision is made clear by the opinion itself. We are thus brought to a consideration of the effect on the present suit of the judgment of the Supreme Court of Idaho.
would formally constitute res judicata in the courts of the state, since it in fact satisfies the requirements of prior adjudication, and since the constitutional issue as to jurisdiction might have been presented to the state supreme court and reviewed here, the decision is a bar to the present suit insofar as it seeks to enjoin the enforcement of the judgment for want of jurisdiction. Compare Fidelity Natl. Bank & Trust Co. v. Swope, 274 U. S. 123, 274 U. S. 130-131.
Third. The surety company contends in No. 21 that, even if the trial court of the state had jurisdiction, the federal District Court may enjoin the enforcement of the judgment on the ground that, having been entered without notice and an opportunity for a hearing on the construction of the bond, it lacked due process of law. It is true that entry of judgment without notice may be a denial of due process even where there is jurisdiction over the person and subject matter. But that rule is not applicable here, for, if the bond, properly construed, stayed the judgment as against Anderson, the surety company consented to the entry of judgment against it without notice for his failure to pay. If the bond did not stay the judgment as against Anderson, the trial court confessedly erred in entering the judgment on the bond. In order to contest its liability, the surety company had the constitutional right to be heard at some time on the construction of the bond. The state practice provided the opportunity for such a hearing by an appeal after the entry of judgment.
In No. 3, writ of certiorari dismissed.
In No. 21, decree reversed.
* Together with No. 21, Baldwin et al. v. American Surety Co., certiorari to the Circuit Court of Appeals for the Ninth Circuit.
"VIVIAN F. BALDWIN and E. R. BALDWIN, Plaintiffs v. SINGER SHOWING MACHINE COMPANY, a Corporation, and ED. ANDERSON, Defendants."
"Whereas, the defendant, Singer Sewing Machine Company, a corporation, in the above entitled action, has appealed to the Supreme Court of the State of Idaho from the judgment made and entered against it in the above entitled action and in the above entitled court in favor of the plaintiffs in said action on the 31st day of May, 1928, for the sum of Nineteen Thousand Five Hundred ($19,500.00) Dollars and for Seventy-three and 70/100 ($73.70) Dollars costs in said suit, making a total of Nineteen Thousand Five Hundred and Seventy-three and 70/100 ($19,573.70) Dollars, and from the whole of said judgment;"
"And whereas, the said appellant, Singer Sewing Machine Company, a corporation, is desirous of staying the execution of said judgment so appealed from;"
"Now therefore the undersigned American Surety Company, a corporation authorized to, and doing business in the Idaho, in consideration of the premises and of such appeal on the part of said appellant, Singer Sewing Machine Company, a corporation, does hereby acknowledge itself firmly bound in the sum of Twenty-five Thousand ($25,000.00) Dollars, gold coin of the United states, that, if the said judgment appealed from, or any part thereof, be affirmed, or the appeal dismissed, the appellant will pay in gold coin of the United states of America the amount directed to be paid as to which said judgment shall be affirmed, if affirmed only in part, and all damages and costs which may be awarded against the appellant upon the appeal, and that, if the said appellant does not make such payment within thirty days from the filing of the remittitur from the Supreme Court in the court from which the appeal is taken, judgment may be entered on motion of the respondents in their favor and against the undersigned surety for the said sum of Nineteen Thousand Five Hundred Seventy-three and 70/100 ($19,573.70) Dollars, together with the interest that may be due thereon and the damages and costs which may be awarded against the said appellant, Singer Sewing Machine Company, upon the appeal."
"In Witness Whereof the said American Surety Company, has caused its name and seal to be attached hereto by its proper officers and agents at Boise, Idaho, this 28th day of August, 1928."
The Idaho Statute was so construed by the Circuit Court of Appeals for the Ninth Circuit in United States Fidel. & Guar. Co. v. Fort Misery Highway Dist., 22 F.2d 369, 373, and in Empire State-Idaho Mining & Developing Co. v. Hanley, 136 F. 99. See also Calif.Code Civ.Proc. § 942; Meredith v. Santa Clara Mining Assn. of Baltimore, 60 Cal. 617, 619; Hitchcock v. Caruthers, 100 Cal. 100, 103; Hawley v. Gray Bros. Artificial Stone Paving Co., 127 Cal. 560, 561. The California provision was the prototype for the Idaho statute in question. See Naylor & Norlin v. Lewiston & S.E. Elec. Ry. Co., 14 Idaho 722, 725. Compare Hartford Accident & Indemnity Co. v. Bunn, 285 U. S. 169; Capital National Bank v. Board of Supervisors, 286 U.S. 550; Fidelity Union Casualty Co. v. Hanson, 287 U.S. 599; Louisville & Nashville R. Co. v. Parker, 287 U.S. 569; Toledo Scale Co. v. Computing Scale Co., 281 F. 488, aff'd, 261 U. S. 261 U.S. 399.
The petition to this Court for a writ of certiorari, although filed October 30, 1931, was not granted until April 25, 1932, 286 U.S. 536, action thereon being withheld "awaiting the action of the Supreme Court of Idaho in the matters pending before it." The actions referred to were two further steps taken by the surety company in the Idaho courts to be relieved of the original judgment against it. The first was a motion to correct, amend, and vacate the original judgment. This motion the trial court overruled, and its order was upheld on appeal to the Supreme Court of Idaho. 13 P.2d 650, decided July 12, 1932, rehearing denied, September 10, 1932. The second was a direct appeal to the Supreme Court of Idaho from the original judgment; this appeal was dismissed because taken more than 90 days after the entry of the judgment appealed from. 51 Idaho 614, decided February 21, 1932.
In Wells Fargo & Co. v. Taylor, 254 U. S. 175, the petitioner had not been allowed to become a party to the prior litigation in the state court.
"In this state, the appellant had a choice between two remedies, and he chose to file his motion to vacate the judgment in the case in which the judgment was rendered upon the same facts as pleaded in the complaint in the action involved, and the court, after hearing the motion, decided the facts against the appellant, holding that the judgment was not void, and the order so holding was appealable. But appellant refused to exercise his right of appeal, and brought this suit in equity to enjoin the collection of said judgment. He had his day in court in that action, and the decision of that motion upon the question of jurisdiction was res adjudicata. The appellant had the right either to attack said judgment by motion in the original case, or, by bringing this action, to enjoin or to have it set aside. If he proceeded by motion and the court decided against him, the decision of that question, until reversed upon appeal, is final and binding on the parties."
Compare the effect, under Idaho law, of a decision on a motion to set aside a judgment because of the mistake, inadvertence, or excusable neglect of the defendant, or to allow an answer to the merits to be interposed after judgment where summons was not served personally on the defendant. Motions of this kind are allowed by express statute. Idaho Comp.Stat. § 6726. They present a matter for judicial discretion, Mortgage Co. Holland America v. Yost, 39 Idaho 489, and their determination does not bar a renewal motion. See Dellwo v. Petersen, 34 Idaho 697. But motions of this kind are to be distinguished from those attacking the judgment as void for want of jurisdiction. Armitage v. Horseshoe Bend Co., Ltd., 35 Idaho 179; Shumake v. Shumake, 17 Idaho 649.
The cases are many in which failure to comply with state rules of practice has prevented this Court from considering a federal claim on direct review. See, e.g., cases where the claim was not considered by the highest court of the state because it was not raised by the proper procedure, Brown v. Massachusetts, 144 U. S. 573, 144 U. S. 580; Hulbert v. Chicago, 202 U. S. 275, 202 U. S. 281; or by the proper pleadings, Atlantic Coast Line R. v. Mims, 242 U. S. 532, 242 U. S. 535-537; Nevada-California-Oregon Ry. v. Burrus, 244 U. S. 103, 244 U. S. 104-105; or was not raised at the proper stage of the proceedings, Spies v. Illinois, 123 U. S. 131, 123 U. S. 181; Baldwin v. Kansas, 129 U. S. 52, 129 U. S. 56-57; Jacobi v. Alabama, 187 U. S. 133; Layton v. Missouri, 187 U. S. 356; Louisville & Nashville R. Co. v. Woodford, 234 U. S. 46, 234 U. S. 51; Missouri Pacific Ry. Co. v. Taber, 244 U. S. 200, 244 U. S. 201-202; Missouri, Kansas & Texas Ry. Co. v. Sealy, 248 U. S. 363, 248 U. S. 365; Barbour v. Georgia, 249 U. S. 454, 249 U. S. 460; Hartford Life Ins. Co. v. Johnson, 249 U. S. 490, 249 U. S. 493-494. Compare Michigan Central R. Co. v. Mix, 278 U. S. 492, 278 U. S. 496.


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Forty-two episodes of Vibrio parahaemolyticus infections were detected in Beira, Mozambique, from January to May 2004. The majority of the isolates (81%) belonged to the pandemic serovars (O3:K6 and O4:K68) of V. parahaemolyticus. The pandemic serovars were positive by group-specific PCR (GS-PCR) and a PCR specific for open reading frame ORF8 (ORF8-PCR), which are molecular markers of the pandemic clone, and were positive for tdh but negative for trh. The remaining 19% of the strains also possessed the tdh gene but were GS-PCR and ORF8-PCR negative and did not belong to the pandemic serovars. Patients with V. parahaemolyticus infection were older (mean age, 27 years) than patients infected by other diarrheal agents (mean age, 21 years). Ten percent of diarrhea patients from whom no V. parahaemolyticus was cultured were severely dehydrated, but none of the V. parahaemolyticus cases were severely dehydrated. This is the first report of the isolation of pandemic strains of V. parahaemolyticus in sub-Saharan Africa and clearly indicates that the pandemic of V. parahaemolyticus has spread into the African continent.
Vibrio parahaemolyticus is a seafood-borne pathogen which can cause gastroenteritis in humans. They are gram-negative, halophilic bacteria that inhabit marine and estuarine environments. V. parahaemolyticus was first isolated in Japan in 1950 as a cause of food-borne illness. This organism is associated with bacterial gastroenteritis in the United States (8, 15) and Europe, and it is one of the most important food-borne pathogens in Asia, causing approximately half of the food-poisoning outbreaks in Taiwan, Japan, Vietnam, and southeast Asian countries (3, 5). To date, 75 different combinations of O and K serotypes of V. parahaemolyticus are recognized (10, 11). All V. parahaemolyticus strains carrying the tdh gene, which encodes thermostable direct hemolysin (TDH), or the trh gene, which encodes TDH-related hemolysin, or both genes, can cause gastroenteritis in humans (17). Environmental strains of V. parahaemolyticus rarely possess either of these genes.
Generally, gastroenteritis caused by V. parahaemolyticus is a multiserovar affliction. However, from February 1996 an abrupt increase in the incidence of V. parahaemolyticus was recorded, and all the strains were identified as belonging to serovar O3:K6 (19). This clone carried the tdh gene but lacked the trh gene and had a unique arbitrarily primed PCR pattern (14, 19). In subsequent years, V. parahaemolyticus isolates belonging to this serovar caused several outbreaks in five Asian countries, Canada, the United States, and Russia. The rapid spread of the O3:K6 strains to different countries after 1996 marked the beginning of the first pandemic of V. parahaemolyticus (14). This was the first occurrence of a single serotype-associated spread of V. parahaemolyticus. The newly recognized serovar also had seven base changes in the toxRS operon. This polymorphism in the toxRS operon was exploited to develop a group-specific PCR (GS-PCR), which has been used as a molecular marker for the identification of the pandemic clone of V. parahaemolyticus (14). The gene coding the filamentous phage f237 is unique for the pandemic V. parahaemolyticus clone (4, 16). One of the open reading frames (ORFs) of the f237 phage genome, ORF8, was proposed as a marker of the pandemic clone (10, 16). However, GS-PCR-positive strains lacking ORF8 were recently reported (3, 13) among the pandemic strains.
Increased rates of isolation of the pandemic serovars of V. parahaemolyticus in Asia and the United States since 1996 have suggested a V. parahaemolyticus pandemic (14, 18, 20, 21). Besides O3:K6, it has been reported that 10 other serotypes (O4:K68, O1:K25, O1:K41, O1:KUT, O1:K56, O3:K75, O4:K8, O4:K12, O4:KUT, and O5:KUT, where UT indicates untypeable) have emerged and have been shown to belong to the pandemic clone by molecular typing techniques (5, 13, 22). Molecular analysis of the pandemic strains indicated that some of these serotypes, especially O4:K68 and O1:KUT, might have diverged from the pandemic strain O3:K6 by alteration of the O and K antigens and by serovar transition (5, 6).
There is no information on V. parahaemolyticus infections in Mozambique or sub-Saharan Africa. During surveillance for cholera in Beira, Mozambique, from December 2003 to January 2004, a large number of V. parahaemolyticus isolates were detected. In this study, we describe the microbiological and preliminary epidemiological aspects of V. parahaemolyticus infections in Beira, Mozambique.
Study site and population.The port city of Beira, located in Sofala Province, is the second largest city in Mozambique, after Maputo. Beira was built on swampy grounds at the mouth of Pungwe River and has a population of approximately 450,000, divided into 22 neighborhoods (bairros). Many areas of Beira are located below sea level. Cholera is endemic in Beira, with cases usually detected from January to June during the rainy season (7). The characteristic marshy areas with brackish water, periodic flooding during the rainy season, the common practice of defecation in open areas, the presence of nontight latrines, and the drainage of municipal waste into the embankments maintain the risk for diarrheal diseases in the area (1). The Cholera Treatment Center is the only site in Beira for the treatment of severe watery diarrhea. Cases presenting to other health care facilities are transferred to the Cholera Treatment Center.
Surveillance.The port city of Beira has a population of approximately 450,000, divided into 22 districts (bairros). The source population for the cases and the controls was the residents of Esturro (a district in the center of Beira). Patients who presented to the Cholera Treatment Center with a history of acute, nonbloody diarrhea from 1 January and 31 May 2004 were eligible as a case if they fulfilled the following criteria: (i) consent or, in the case of minors, guardian or parental consent to participate in the study; (ii) a resident of Esturro since 11 December 2003; (iii) not pregnant, if female; and (iv) at least 2 years of age during the time of the mass vaccination. After the initial medical assessment and rehydration were completed, a case report form was completed and a stool specimen was collected from all enrolled cases. The case report form included questions on demographics, medical history, treatment, environmental and socioeconomic factors, and information about oral cholera vaccination. By using the address and directions obtained at the Cholera Treatment Center, each case's household was located to verify residence in Esturro and to check his or her vaccination card (if it was not available during presentation at the Cholera Treatment Center).
Stool specimens or rectal swabs were collected from Esturro residents over 2 years of age presenting to the Cholera Treatment Center with acute nonbloody diarrhea. In addition one specimen was collected daily from a patient residing in a bairro other than Esturro. The clinical features of all diarrhea patients were recorded on standardized case report forms. Severe dehydration was defined according to World Health Organization guidelines based on clinical presentation (24).
Stool culture.The nonbloody stool samples or rectal swabs (RS) were collected in Cary-Blair transport medium and transported at ambient temperature to the laboratory within 2 h. The stool samples or RS were plated directly on the thiosulfate citrate bile salt sucrose (TCBS) agar (Eiken, Japan) and taurocholate tellurite gelatine agar (TTGA). The specimens were also enriched in alkaline peptone water for 6 h (pH 8.6, 37°C) (23) and then plated on TCBS agar and TTGA. Sucrose-nonfermenting green colonies on the TCBS agar that were oxidase and gelatinase positive and O/129 sensitive on TTGA were initially suspected to be V. parahaemolyticus and were stored in Luria agar slants with 3% salt for biochemical identification. Most of the suspected V. parahaemolyticus isolates grew as pure cultures on TTGA and TCBS agar plates with the characteristic colony morphology on the culture plates. Confirmation of the isolates as V. parahaemolyticus was done by standard methods (12).
O and K serotyping.The O (somatic) and K (capsular) antigen typing of V. parahaemolyticus isolates was done by using commercial antisera, according to the protocol and instructions described by the manufacturer (Toshiba Kagaku Kogyo Co., Ltd., Tokyo, Japan).
PCR assays.PCR assays were performed to detect the toxR, tdh, and trh genes of V. parahaemolyticus by using the primers described previously (5, 9, 12). GS-PCR and a PCR for ORF8 (ORF8-PCR) were performed to detect molecular markers of the pandemic clone (5, 13, 14). Template DNA was prepared by growing the bacteria in Luria broth (Difco, Detroit, Mich.) with 3% NaCl at 37°C overnight, centrifuging the culture, resuspending the pellet in sterile distilled water, and boiling for 10 min. The PCR products were electrophoresed in a 1% agarose gel stained in ethidium bromide and visualized under UV light with a transilluminator.
Antimicrobial susceptibility test.Antimicrobial susceptibility testing was done by the Kirby-Bauer disk diffusion method (2) with control strain Escherichia coli ATCC 25922.
Ethical considerations.This project was approved by the government of Mozambique; the Institutional Review Board of the International Vaccine Institute, Seoul, Korea; and the Secretariat Committee on Research Involving Human Subjects of the World Health Organization, Geneva, Switzerland. Informed consent was obtained verbally at the community level through meetings with community leaders of Beira. Individual written consent was obtained from all cases prior to their participation in the study.
A total of 5,128 diarrhea episodes were treated at the Cholera Treatment Center from 1 January to 31 May 2004. Stool specimens or rectal swabs were collected from 403 diarrhea patients from Esturro. V. parahaemolyticus was isolated from 42 stool samples between 24 February and 31 May 2004; 32 of the 42 isolates belonged to serovar O3:K6, and 2 isolates belonged to O4:K68. Seven isolates belonged to serovar O3:K58, and one isolate belonged to O4:K13. All strains of O3:K6 and O4:K68 were positive for toxR and tdh and were positive by GS-PCR and orf8-PCR (Table 1). The serovar O3:K58 and O4:K13 isolates were positive for toxR and tdh but were negative for trh, GS-PCR, and orf8-PCR (Table 1).
As shown in Table 2, the V. parahaemolyticus-infected patients were older than the other diarrhea patients. None of the 42 patients infected by V. parahaemolyticus was under 5 years of age, but 55 of the 361 patients (15%) from whose stool specimens no V. parahaemolyticus was isolated were under 5 years of age (P = 0.007) (Table 2). Six of 42 of the V. parahaemolyticus-infected patients (15%) required intravenous rehydration, whereas 47% (170 of 361) of the patients from whom no V. parahaemolyticus was isolated required intravenous rehydration (P < 0.001). Similar percentages of V. parahaemolyticus patients (81%) and diarrhea patients from whom V. parahaemolyticus could not be isolated (74%) presented with vomiting (P = 0.3). None of the patients was coinfected with V. parahaemolyticus and V. cholerae.
All the strains of serovars O3:K58, O4K68, and O4:K13 were susceptible to tetracycline, ampicillin, sulfamethoxazole-trimethoprim, nalidixic acid, furazolidone, erythromycin, and ciprofloxacin, whereas all strains of serovar O3:K6 were resistant to ampicillin but susceptible to all other antibiotics. According to both serological and molecular markers, all strains of serovars O3:K6 and O4:K68 belonged to the pandemic genotype (Table 1).
The large number of V. parahaemolyticus infections during a diarrhea surveillance study in Beira, Mozambique, was a serendipitous finding. The majority (81%) of the strains isolated in the study belonged to serovars O3:K6 and O4:K68 and therefore belonged to the pandemic serogroup. In Asia and the United States, the pandemic strains of V. parahaemolyticus have been detected since 1996. This is the first report on the isolation of pandemic serovars of V. parahaemolyticus in the African continent. There is therefore a need to consider V. parahaemolyticus in the differential diagnosis of etiologic agents for watery diarrhea in individuals residing in Africa or coming from Africa.
Our study had several limitations: children under 2 years were not tested, and surveillance was treatment center based and thereby tended to reflect more severe diarrheal cases in the community. The Cholera Treatment Center was known to the community as a place for the treatment of watery diarrhea; thus, our surveillance may have missed diarrhea cases with nonwatery presentations. The only other enteropathogen tested for in the surveillance was V. cholerae; thus, coinfections with V. parahaemolyticus and diarrheal agents other than V. cholerae may have been more common than our data would suggest. A similar number of patients with V. parahaemolyticus and diarrhea caused by other agents presented with vomiting, but severe dehydration was less frequently seen in the V. parahaemolyticus-infected patients. This finding may be related to the fact that V. parahaemolyticus-infected patients were older than patients with other causes of diarrhea. None of the V. parahaemolyticus patients was under 5 years of age. The absence of V. parahaemolyticus in patients under 5 years of age is striking and may account for the significant differences in rehydration, and we plan to study this in the future.
This finding may point toward a potential route of transmission. Tuyet and coworkers (21) have described the potential role of the consumption of uncooked or partially cooked seafood in the transmission of V. parahaemolyticus in Vietnam. It seems possible that older individuals are at higher risk than younger children for V. parahaemolyticus infection because of different exposures. In contrast to Far East Asia, raw seafood is not considered a delicacy in Beira. However, dried shrimp is frequently eaten uncooked, especially in the absence of cooking facilities. Shellfish are a popular source of protein but tend to be eaten cooked. Secondary contamination of foods in kitchens is likely to be a potential route of transmission in settings like this where raw seafood is not consumed.
The focus of our surveillance was on Esturro, a bairro with a mixed labor force. People from rural areas moved into this slum during the civil war, which ended in 1992, because of the higher level of safety in urban areas than in isolated areas. The infrastructure, including the water supply and sanitation, has remained at a minimal level. Outside Esturro, many residents frequently earn a living through agricultural activities in the countryside. Little if any care is paid to food safety. The water supply and sanitation have remained rudimentary in many households. It seems likely that increased attention to hygiene and food safety should reduce the incidence of V. parahaemolyticus infections. However, before recommendations on the means of decreasing the risk of V. parahaemolyticus infections can be made, a better understanding of the risk factors is needed. Such studies should include individuals from all parts of Beira, as the risk of infection may differ between bairros.
We are grateful for the enthusiastic support that we received from our Mozambiquean collaborators. We thank Catarina Mondlane and Raul Vaz for laboratory work.
This work was supported by the Diseases of the Most Impoverished Program, funded by the Bill and Melinda Gates Foundation and coordinated by the International Vaccine Institute. ICDDR,B is supported by the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Japan, Kingdom of Saudi Arabia, The Netherlands, Sweden, Sri Lanka, Switzerland, and the United States.
Returned for modification 5 January 2005.
Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turk. 1966. Antibiotic susceptibility testing by standardized single disk method. Am. J. Clin. Pathol. 145:493-496.
Chang, B., S. Yoshida, H. Miyamoto, M. Ogawa, K. Horikawa, K. Ogata, M. Nishibuchi, and H. Taniguchi. 2000. A unique and common restriction fragment pattern of the nucleotide sequences homologous to the genome of vf33, a filamentous bacteriophage, in pandemic strains of Vibrio parahaemolyticus O3:K6, O4:K68, and O1:K untypeable. FEMS Microbiol. Lett. 192:231-236.
Chowdhury, A., M. Ishibashi, V. D. Thiem, D. T. Tuyet, T. V. Tung, B. T. Chien, L. Seidlein Lv, G. Canh do, J. Clemens, D. D. Trach, and M. Nishibuchi. 2004. Emergence and serovar transition of Vibrio parahaemolyticus pandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and 1999. Microbiol. Immunol. 48:319-327.
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Ishibashi, M., K. Ohta, T. Shimada, T. Honda, J. Sugiyama, T. Miwatani, and H. Yokoo. 2000. Current status of OK serotype combinations of Vibrio parahaemolyticus. Nippon Saikingaku Zasshi 55:535-541.
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Tuyet, D. T., V. D. Thiem, L. Von Seidlein, A. Chowdhury, E. Park, G. Canh do, B. T. Chien, T. Van Tung, A. Naficy, M. R. Rao, M. Ali, H. Lee, T. H. Sy, M. Nichibuchi, J. Clemens, and D. D. Trach. 2002. Clinical, epidemiological, and socioeconomic analysis of an outbreak of Vibrio parahaemolyticus in Khanh Hoa Province, Vietnam. J. Infect. Dis. 186:1615-1620.
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