Evaluating the massive underreporting and undertesting of COVID-19 cases in multiple global epicenters.

BACKGROUND: With continuous global COVID-19 outbreak, differing case numbers and mortality rates are observed. While actual case numbers appear vague, mortality numbers related to COVID-19 seem more precise. In this study, we used the mortality rate as the main indicator to evaluate the extent of underreporting and underdetection of COVID-19 cases. METHODS: We have analyzed all available data provided by the World Health Organization on the development of international COVID-19 cases and mortality numbers on March 17th, 2020. A crude case-fatality risk (cCFR) and adjusted case-fatality risk (aCFR) was calculated for China, South Korea, Japan, Italy, France, Spain, Germany, Iran and the United States. Additionally, a fold-change (FC) was derived for each country. RESULTS: The highest aCFR and FC were detected for Spain. Based on their FC values, an extremely high number of undetected COVID-19 cases was displayed in France, the United States, Italy and Spain. For these countries, our findings indicate a detection rate of only 1-2% of total actual COVID-19 cases. CONCLUSIONS: Due to limited testing capacities, mortality numbers may serve as a better indicator for COVID-19 case spread in many countries. Our data indicate that countries like France, Italy, the United States, Iran and Spain have extremely high numbers of undetected and underreported cases. Differences in testing availability and capacity, containment as well as overall health care and medical infrastructure result in significantly different mortality rates and COVID-19 case numbers for each respective country.

Anti-inflammatory properties and expression in selected organs of canine interleukin-1ß splice variant 1.

The IL-1ß gene can be also be spliced with the intron 4 retention; the result is a IL-1ß splice variant 1 (IL-1ßsv1), which was significantly up-regulated in failing myocardium of dogs suffering from chronic degenerative valvular disease (CDVD). Expression of IL-1ßsv1 was assessed, at both RNA and protein levels, in organs affected by heart failure, namely, kidneys, liver, and lungs from 35 dogs suffering chronic degenerative valvular disease (CDVD) and in 20 disease free control dogs. IL-1ßsv1 RNA was detected in the dogs from both groups. In the CDVD group, the highest RNA and protein IL-1ßsv1 levels were observed in lungs, followed, in that order, by the liver and kidneys. IL-1ßsv1 protein was found in the cytoplasm of hepatocytes and IL-1ßsv1-overexpressing DH82 cells. In lungs, IL-1ßsv1 was localized in the cytoplasm and in the nuclei of bronchiolar epithelial and smooth-muscle cells. Cytoplasmic and nuclear IL-1ßsv1 expression was observed in macrophages, and a strong nuclear signal was detected in epithelial cells of the alveolar sacs. Following lipopolysaccharide (LPS) stimulation, overexpression of IL-1ßsv1 in DH82 cells decreased the pro-inflammatory response. Our results indicate that IL-1ßsv1 is constitutively expressed in both normal tissues and in tissues from cases of heart failure. The presence of IL-1ßsv1 in tissues exposed to invading agents and its anti-inflammatory activity in DH82 cells may point to its immunomodulatory role in vivo.

Prevalence and antimicrobial resistance of Arcobacter butzleri and Arcobacter cryaerophilus isolates from retail meat in Lower Silesia region, Poland.

Arcobacter butzleri and A. cryaerophilus are considered potential foodborne pathogens. Consumption of Arcobacter-contaminated food is regarded the most likely source of human poisoning. We investigated the prevalence and antimicrobial resistance of Arcobacter isolates in 210 retail meat samples. Seventy-nine A. butzleri and 6 A. cryaerophilus were isolated from pork, beef and chicken meat. Incidence ofA. butzleri was found to be the highest in chicken meat (83%). Less of A. butzleri was isolated from beef (16%) and pork (14%). Most of the A. butzleri isolates were resistant to ß-lactams, like ampicillin (85%), amoxicillin with clavulonic acid (63%), cefotaxime (66%) and mac- rolides, i.e., erythromycin (62%). In contrast, all except one A. cryaerophilus isolates were susceptible to erythromycin. Tetracycline and aminoglycosides showed the highest efficacy against A. butzleri and A. cryaerophilus since almost 80% of their population was susceptible to these agents. All, except one A. cryaerophilus and the majority ofA. butzleri isolates (70%) were susceptible to fluoroquinolones. The incidence of multiresistant isolates was found in forty two (53%) A. butzleri, and one (16%) A. cryaerophilus isolates Eight A. butzleri isolates were resistant to all antimicrobials tested. These results indicate significant incidence of potential foodborne zoonotic agents, i.e. A. butzleri and A. cryaerophilus including multiresistant isolates in retail meat in Poland.

Increased gene expression of catecholamine-synthesizing enzymes in adrenal glands contributes to high circulating catecholamines in pigs with tachycardia-induced cardiomyopathy.

High levels of circulating catecholamines have been established as fundamental pathophysiological elements of heart failure (HF). However, it is unclear whether the increased gene expression of catecholamine-synthesis enzymes in the adrenal glands contributes to these hormone abnormalities in large animal HF models. We analyzed the mRNA levels of catecholamine-synthesizing enzymes: tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD), dopamine-ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands of 18 pigs with chronic systolic non-ischaemic HF (tachycardia-induced cardiomyopathy due to right ventricle pacing) and 6 sham-operated controls. Pigs with severe HF demonstrated an increased expression of TH and DBH (but neither AAAD nor PNMT) as compared to animals with milder HF and controls (P<0.05 in all cases). The increased adrenal mRNA expression of TH and DBH was accompanied by a reduced left ventricle ejection fraction (LVEF) (P<0.001) and an elevated plasma B-type natriuretic peptide (BNP) (P<0.01), the other indices reflecting HF severity. There was a positive relationship between the increased adrenal mRNA expression of TH and DBH, and the high levels of circulating adrenaline and noradrenaline (all P<0.05). The association with noradrenaline remained significant also when adjusted for LVEF and plasma BNP, suggesting a significant contribution of adrenals to the circulating pool of catecholamines in subjects with systolic HF.

Characteristics of the bacterial flora in the conjunctival sac of cats from Poland.

OBJECTIVES: To assess the bacterial flora of the conjunctival sac in clinically healthy cats and cats with signs of conjunctivitis. METHODS: A total of 324 conjunctival swabs were examined between 2011 and 2012 taken from 60 animals, 30 of which were clinically healthy and 30 with signs of chronic conjunctivitis. The samples were taken three times at 4-week intervals from the clinically healthy cats. The samples from the cats with conjunctivitis were taken before and 4 weeks after cessation of successful therapy. Swabs from both the right and left eye of each cat were subjected to microbiological examination and polymerase chain reaction for the presence of DNA of Chlamydophila felis and Mycoplasma felis. RESULTS: There was no qualitative difference in the eye microflora between the clinically healthy animals and those with signs of conjunctivitis. Staphylococcus epidermidis (21 · 9%) was the most common microorganism isolated and it was more commonly detected in swabs from cats with conjunctivitis (P < 0 · 0001) as was Staphylococcus aureus (P = 0 · 07). The presence of C. felis was significantly correlated with (P < 0 · 0001) signs of conjunctivitis and was detected in 25% of swabs collected from both conjunctival sacs. No DNA of M. felis was detected in any swab. None of the animals had sterile conjunctival sacs in all consecutive bacteriological tests. CLINICAL SIGNIFICANCE: The conjunctival sac in cats was sterile in over 50% of the clinically healthy cats and 25% of the cats with conjunctivitis. The sterility did not persist for longer than 4 weeks. Positive bacterial cultures occur in cats with and without clinical signs of conjunctivitis.

Invasiveness of Listeria monocytogenes strains isolated from animals in Poland.

Animals are important reservoir of Listeria monocytogenes, a pathogen causing serious infections in both humans and livestock. However, data on invasiveness of L. monocytogenes strains of animal origin is very scarce. Ability of 18 L. monocytogenes strains of animal origin to invade HT-29 cells was investigated. Plaque forming assay was used to assess invasiveness and ability of the pathogen to spread in the cell line. Almost 40% of L. monocytogenes strains were weakly invasive. It was shown that strains from serogroup 4b exhibited the highest invasiveness, whereas serogroup 1/2b consisted of strains of invasiveness below 0.0001%. Analysis of translated inlA and inlB gene sequences revealed no premature stop codons. Lineage-specific mutations in low invasive strains were identified within inlA and inlB sequences. Our results demonstrate high incidence of low invasive animal L. monocytogenes strains, which may be at least partly explained by unique point mutations in the InlA and InlB.

Genotypes and oxacillin resistance of Staphylococcus aureus from chicken and chicken meat in Poland.

The genotypes and oxacillin resistance of 263 Staphylococcus aureus isolates cultured from chicken cloacae (n = 138) and chicken meat (n = 125) was analyzed. Fifteen spa types were determined in the studied S. aureus population. Among 5 staphylococcal protein A gene (spa) types detected in S. aureus from chicken, t002, t3478, and t13620 were the most frequent. Staphylococcus aureus isolates from meat were assigned to 14 spa types. Among them, the genotypes t002, t056, t091, t3478, and t13620 were dominant. Except for 4 chicken S. aureus isolates belonging to CC398, the remaining 134 isolates were clustered into multilocus sequence clonal complex (CC) 5. Most of meat-derived isolates were assigned to CC5, CC7, and CC15, and to the newly described spa-CC12954 complex belonging to CC1. Except for t011 (CC398), all other spa types found among chicken isolates were also present in isolates from meat. Four S. aureus isolated from chicken and one from meat were identified as methicillin-resistant S. aureus (MRSA) with oxacillin minimum inhibitory concentrations from 16 to 64 µg/mL. All MRSA were assigned to spa types belonging to ST398, and included 4 animal spa t011 SCCmecV isolates and 1 meat-derived spa t899, SCCmecIV isolate. Borderline oxacillin-resistant S. aureus (BORSA) isolates, shown to grow on plates containing 2 to 3 µg/mL of oxacillin, were found within S. aureus isolates from chicken (3 isolates) and from meat (19 isolates). The spa t091 and t084 dominated among BORSA from chicken meat, whereas t548 and t002 were found within animal BORSA. We report for the first time the presence of MRSA in chicken in Poland. We demonstrate that MRSA CC398 could be found in chicken meat indicating potential of introduction of animal-associated genotypes into the food chain. We also report for the first time the possibility of transmission of BORSA isolates from chicken to meat.

Matrix metalloproteinase 9/neutrophil gelatinase associated lipocalin/tissue inhibitor of metalloproteinasess type 1 complexes are localized within cardiomyocytes and serve as a reservoir of active metalloproteinase in porcine female myocardium.

Matrix metalloproteinase 9 (MMP-9) is crucial for physiological tissue repair and pathophysiological myocardial remodeling. The regulation of its functioning has been shown to be mediated by formation of complexes with tissue inhibitor of metalloproteinases 1 (TIMP-1) and neutrophil gelatinase associated lipocalin (NGAL). We investigated the mRNA and protein expression of MMP-9, TIMP-1 and NGAL, the formation of complexes, their gelatinolytic activity and cellular localization in left ventricle (LV) from 10 female pigs with induced systolic heart failure (HF), 5 control pigs, and a woman with severe HF. The MMP-9, TIMP-1 and NGAL mRNA in LV did not differ between diseased and healthy pigs. In all pigs MMP-9, TIMP-1 and NGAL proteins were present in LV as high molecular weight (HMW) complexes (115, 130, 170 and 220 kDa), and no monomers were found. A 80 and 115 kDa gelatinolytically active bands were present in all LV homogenates. A 130-kDa active band was seen only in LV from pigs with severe HF. Similar results were found in the explanted heart of a female patient with severe HF. The incubation of the homogenates of porcine LV at 37°C resulted in appearance of 88 kDa active band, which was accompanied by a decreased intensity of HMW bands. The incubation of the homogenates of porcine LV (depleted of active MMP-9) with trypsin generated 80 and 115 kDa active bands. Immunohistochemistry revealed the presence of MMP-9 in the cytoplasm of porcine cardiomyocytes, but not in cardiofibroblasts. Our data suggest that MMP-9 originates from cardiomyocytes, forms the gelatinolytically inactive complexes with TIMP-1 and NGAL, present in normal and failing myocardium, likely serving as a reservoir of active MMP-9. Further studies are needed to elucidate the role of these HMW complexes in the extracellular matrix remodeling during the progression of HF, which presence should be considered when developing efficient strategies inhibiting myocardial matrix metalloproteinases.

Effect of short-term rapid ventricular pacing followed by pacing interruption on arterial blood pressure in healthy pigs and pigs with tachycardiomyopathy.

Ventricular tachycardia may lead to haemodynamic deterioration and, in the case of long term persistence, is associated with the development of tachycardiomyopathy. The effect of ventricular tachycardia on haemodynamics in individuals with tachycardiomyopathy, but being in sinus rhythm has not been studied. Rapid ventricular pacing is a model of ventricular tachycardia. The aim of this study was to determine the effect of rapid ventricular pacing on blood pressure in healthy animals and those with tachycardiomyopathy. A total of 66 animals were studied: 32 in the control group and 34 in the study group. The results of two groups of examinations were compared: the first performed in healthy animals (133 examinations) and the second performed in animals paced for at least one month (77 examinations). Blood pressure measurements were taken during chronic pacing--20 min after onset of general anaesthesia, in baseline conditions (20 min after pacing cessation or 20 min after onset of general anaesthesia in healthy animals) and immediately after short-term rapid pacing. In baseline conditions significantly higher systolic and diastolic blood pressure was found in healthy animals than in those with tachycardiomyopathy. During an event of rapid ventricular pacing, a significant decrease in systolic and diastolic blood pressure was found in both groups of animals. In the group of chronically paced animals the blood pressure was lower just after restarting ventricular pacing than during chronic pacing. Cardiovascular adaptation to ventricular tachycardia develops with the length of its duration. Relapse of ventricular tachycardia leads to a blood pressure decrease more pronounced than during chronic ventricular pacing.

Reduction of prevalence of persistent BVDV infection in cattle herds by long-term vaccination program (preliminary clinical study).

Effectiveness of long-term anti-BVDV vaccination program in reducing prevalence of persistent BVDV infection in cattle herds was evaluated in seven years observational study (2005-2011). Among three seropositive dairy cattle herds (within herd seroprevalence 100%, confirmed by ELISA Herd Check BVDV Ab, IDEXX, Sweden) vaccination program based on inactivated vaccine (cytopathic strain 5960) was commenced in 2007 in two herds and continued till 2010. In the years 2007-2011 all calves aged 2-12 weeks in all three herds were tested yearly with RT-PCR in order to detect persistently infected individuals. For the entire study period true prevalence of BVDV persistent infection was significantly lower in vaccinated than in non-vaccinated herd. This may imply the role of long-term vaccination program in reducing prevalence of persistent BVDV infection in cattle herds.

Enterotoxigenic potential of coagulase-negative staphylococci.

Staphylococci are a worldwide cause of human and animal infections including life-threatening cases of bacteraemia, wound infections, pyogenic lesions, and mastitis. Enterotoxins produced by some staphylococcal species were recognized as causative agents of staphylococcal food poisoning (SFP), being also able to interrupt human and animal immune responses. Only enterotoxins produced by Staphylococcus aureus were as yet well characterized. Much less is known about enterotoxigenic potential of coagulase-negative species of genus Staphylococcus (CNS). The pathogenic role of CNS and their enterotoxigenicity in developing SFP has not been well established. Although it has been reported that enterotoxigenic CNS strains have been associated with human and animal infections and food poisoning, most of research lacked a deeper insight into structure of elements encoding CNS enterotoxins. Recent studies provided us with strong evidence for the presence and localization of enterotoxin-coding elements in CNS genomes and production of enterotoxins. Thus, the importance of pathogenic potential of CNS as a source of staphylococcal enterotoxins has been highlighted in human and animal infections as well as in food poisoning.

Salt stress-induced invasiveness of major Listeria monocytogenes serotypes.

UNLABELLED: Of the 13 serotypes, 4b serotype strains are responsible for the majority of recorded invasive listeriosis outbreaks, although some recent listeriosis outbreaks have been attributed to strains of serotypes 1/2a and 1/2b. Virulence and response to osmotic stress in 41 Listeria monocytogenes strains representing serotypes 1/2a, 1/2b and 4b was investigated. It was found that serotype 4b and 1/2b strains exhibited highest invasion efficiency and formed largest plaques in HT-29 cell monolayer. Invasiveness in response to 10-min exposure to 0·3 mol l⁻¹ NaCl was the highest in serotype 4b strains. We demonstrated that 4b serotype L. monocytogenes strains not only have the greatest pathogenic potential but also are the most invasive in response to salt stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Listeria monocytogenes 4b serotype strains are responsible for the majority of recorded invasive listeriosis outbreaks. We showed that strains of serotype 4b are not only the most virulent L. monocytogenes strains but also have the best capacity to enhance their invasiveness in response to salt stress. Our results suggest possession of effective stress response mechanisms of 4b serotype strains, which may contribute to the high infection potential of this subpopulation.

Prevalence of antibiotic resistance genes in staphylococci isolated from ready-to-eat meat products.

Prevalence of mecA, blaZ, tetO/K/M, ermA/B/C, aph, and vanA/B/C/D genes conferring resistance to oxacillin, penicillin, tetracycline, erythromycin, gentamicin, and vancomycin was investigated in 65 staphylococcal isolates belonging to twelve species obtained from ready-to-eat porcine, bovine, and chicken products. All coagulase negative staphylococci (CNS) and S. aureus isolates harbored at least one antibiotic resistance gene. None of the S. aureus possessed more than three genes, while 25% of the CNS isolates harbored at least four genes encoding resistance to clinically used antibiotics. In 15 CNS isolates the mecA gene was detected, while all S. aureus isolates were mecA-negative. We demonstrate that in ready-to-eat food the frequency of CNS harboring multiple antibiotic resistance genes is higher than that of multiple resistant S. aureus, meaning that food can be considered a reservoir of bacteria containing genes potentially contributing to the evolution of antibiotic resistance in staphylococci.

Resistance patterns and occurrence of virulence determinants among GRE strains in southwestern Poland.

PURPOSE: The aim of this study was to determine the antimicrobial resistance and the occurrence of virulence determinants among glycopeptide-resistant enterococci (GRE) isolated in 2007-2009 from patients hospitalized in southwestern Poland. MATERIAL AND METHODS: The minimal inhibitory concentrations (MICs) of antibiotics were determined by agar dilution method or by E-test®. The presence of vanA - vanG resistance and virulence genes (agg, esp, gelE and cylA, cylB, cylM) was investigated using PCR. The ability to form biofilm and the activity of gelatinase, hemolysins, lipase and DNase were tested. RESULTS: All the GRE strains were susceptible to linezolid, daptomycin, and tigecycline and resistant to norfloxacin. In the Enterococcus faecium group, 17 strains carried the vanA gene and 20 the vanB gene. In the Enterococcu faecalis group, 4 strains carried the vanA gene and 1 the vanB gene. There were differences in tetracycline susceptibility between the VanA (70%) and the VanB (55%) phenotypes. Only linezolid had high activity against both the VanA and the VanB phenotypes. The esp gene was present in most of the GRE strains, but only 3 E. faecalis strains produced biofilm. Lipase was produced by 10/42 examined strains, gelatinase by 4/42 and hemolysin by 3/42 isolates. CONCLUSIONS: Linezolid seems to be the optimal option in empirical therapy of infections caused by GRE strains because of the relationship between its activity (MIC value) and susceptibility breakpoint. There was no correlation between the prevalence of different virulence genes and resistance to the antibiotics tested.

Real-time PCR detection of Mycoplasma felis in domestic cats suffering from chronic conjunctivitis (Poland).

Real-time PCR directed to intergenic spacer (IGS) noncoding region between 16S and 23S rRNA genes was used for species specific detection of Mycoplasma felis in conjunctival scrapings. Samples were collected from 57 cats suffering from chronic conjunctivitis in 2008-2010 (Wroclaw, Poland). Samples from 36 cats (63.2%) were shown to be positive for Mycoplasma felis. Our research gives a first insight in the occurrence of Mycoplasma felis among domestic cats in Poland suggesting that this pathogen may constitute an underestimated cause of chronic conjunctivitis.

Genotypic characterization of hospital Enterococcus faecalis strains using multiple-locus variable-number tandem-repeat analysis.

AIMS: The level of genetic diversity and relationships between the specific genotypes and the distribution of virulence determinants among Enterococcus faecalis strains isolated from patients hospitalized in different wards of two hospitals were investigated. METHODS AND RESULTS: Fifty-six clinical strains of E. faecalis, isolated from patients hospitalized in the period of 1999-2004 in several wards in Wroclaw (Poland), were analysed by multiple-locus variable-number tandem-repeat analysis (MLVA). Analysis of seven genomic loci identified 40 novel genotypes among the analysed E. faecalis strains, with two major genomic groups, designated I and II, distinguished at a cut-off of 35%. With a similarity cut-off of 85.7%, the genotypes could be combined into 12 clusters (C1-C12), containing at least two isolates. The remaining 18 MLVA types were represented by a single isolate. CONCLUSIONS: Based on the data obtained by MLVA, it was found that (i) many E. faecalis isolates recovered from patients from the wards whose location allowed the potential transmission of micro-organisms, belonged to closely related MLVA types and (ii) possible relationships between specific E. faecalis genotype and the virulence factors lipase, haemolysin and esp gene can exist. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study confirms that MLVA is a suitable method for the epidemiological study of E. faecalis and for the first time shows possible relationships between specific genotypes and such virulence determinants, i.e. lipase, haemolysin and esp gene.

Subtyping of Listeria monocytogenes isolates by actA gene sequencing, PCR-fingerprinting, and cell-invasion assay.

Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II. A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species.

The impact of environmental stress on Listeria monocytogenes virulence.

Listeria monocytogenes, a significant food-borne pathogen, must defy a variety of conditions encountered in the food environment and during the infection process. In reaction to adverse conditions, the bacteria significantly change their metabolism, inducing a stress response which is mediated by a range of alternative sigma factors. The extent of the response to stress was shown to vary in the L. monocytogenes population. According to recent evidence a major L. monocytogenes alternative sigma factor, designated sigma B (sigma B), regulates some virulence genes in response to stress, which supports an older hypothesis that stress-resistant strains should be more pathogenic. The induction of sigma B-dependent genes may also be important from the point of view of food hygiene. It seems that stress response activation can paradoxically enhance resistance to agents used in food preservation. Therefore, monitoring the expression of sigma B-dependent genes can serve as a useful marker to assess the innate resistance of L. monocytogenes strains. This knowledge will allow the design of new methods with sequential preservation steps that could inactivate the bacteria without inducing their stress response.

Importance of newly detected staphylococcal enterotoxins for food safety.

Staphylococcal enterotoxins (SEs) are emetic toxins causing food poisoning in humans, because of their biological activity and structural relatedness They have been classified as members of the pyrogenic exotoxin superantigen family Among them nine major antigenic types of emetic enterotoxins were recognized In recent years several newly detected SEs were also discriminated, but their occurrence and role in human and animal diseases are not fully understood Neverthless, evidences of their pathogenic role and broad distribution in staphylococcal strains cumulate Therefore their importance as potential risk factor for food safety becomes essential For this reason their properties, genetic determinants and supposed mechanisms of the pathogenic activity are discussed in respect of their potential hazard to human health.

Pancreatic secretory trypsin inhibitor acts as an effective inhibitor of cysteine proteinases gingipains from Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis has been implicated as the major pathogen of periodontitis in adults. This organism produces an array of virulence factors, of which cysteine proteinases, referred to as gingipains K and R, are believed to play a crucial role in pathogenicity. The aim of this study was to investigate the susceptibility of gingipains K and R to inhibition by a pancreatic secretory trypsin inhibitor. MATERIAL AND METHODS: Enzyme activities were measured spectrophotometrically using chromogenic turnover substrates. To estimate the value of the association constant (Ka), constant amounts of enzyme were reacted with increasing amounts of inhibitor to reach equilibrium. The Ka was calculated by fitting the experimental data to the given equation. RESULTS: In this study it was shown that gingipains are susceptible to pancreatic Kazal-type trypsin inhibitors (pancreatic secretory trypsin inhibitors). Bovine pancreatic secretory trypsin inhibitor, having an Arg residue at the P1 position of the reactive site, specifically inhibited the activity of the Arg-specific cysteine proteinase gingipain R, whereas porcine inhibitor, possessing a Lys residue at the P1 position, exhibited activity only against the Lys-specific cysteine proteinase gingipain K. The Ka values for the inhibitor-proteinase interaction were 1.6 x 10(6) m(-1) and 2.0 x 10(4) m(-1) for gingipain R and gingipain K, respectively. CONCLUSION: This finding is the first demonstration of the inhibitory potency of the Kazal-type specific trypsin inhibitors against cysteine proteinases. These discoveries open new possibilities for the use of naturally occurring inhibitors, displaying activity across enzyme families, as a model in designing new molecules of therapeutic significance.