Stenotrophomonas Species Research Articles

Stenotrophomonas ginsengisoli sp. nov., isolated from a ginseng field

A Gram-negative, non-spore-forming, rod-shaped bacterium, designated strain DCY01(T), was isolated from soil from a ginseng field in South Korea and was characterized in order to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that strain DCY01(T) belonged to the Gammaproteobacteria and was most closely related to Stenotrophomonas koreensis KCTC 12211(T) (98.4 % similarity), Stenotrophomonas humi R-32729(T) (97.2 %), Stenotrophomonas terrae R-32768 (97.1 %), Stenotrophomonas maltophilia DSM 50170(T) (96.9 %) and Stenotrophomonas nitritireducens DSM 12575(T) (96.8 %). Chemotaxonomic analyses revealed that strain DCY01(T) possessed a quinone system with Q-8 as the predominant compound, and iso-C(15 : 0) (28.2 %), C(16 : 0) 10-methyl (13.2 %), iso-C(15 : 1) F (10.8 %) and C(15 : 0) (7.5 %) as major fatty acids, corroborating assignment of strain DCY01(T) to the genus Stenotrophomonas. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The results of DNA-DNA hybridization and physiological and biochemical tests clearly demonstrated that strain DCY01(T) represents a species distinct from recognized Stenotrophomonas species. Based on these data, DCY01(T) (=KCTC 12539(T)=NBRC 101154(T)) should be classified as the type strain of a novel species of the genus Stenotrophomonas, for which the name Stenotrophomonas ginsengisoli sp. nov. is proposed.

Risk Factors for Relapse of Ventilator-Associated Pneumonia in Trauma Patients

Our goal was to define risk factors for ventilator-associated pneumonia (VAP) relapse and examine the implications, if any, for initial therapy in trauma patients. Trauma patients cared for in the surgical intensive care unit during a 48-month period with confirmed VAP recurrence were evaluated. Recurrent VAP was defined as a positive quantitative culture (> or = 10(4) colony-forming units/mL in a bronchoalveolar lavage or protected catheter lavage specimen) > or = 4 days after initiation of antibiotics for the primary episode. Recurrence with at least one of the initial causative pathogens was defined as a relapse. Initial causal pathogen, Acute Physiology and Chronic Health Evaluation II score, injury severity score, Glasgow Coma Score (GCS), age, white blood cell count (WBC), and duration of hospital stay before diagnosis were analyzed in univariate and multivariate regression models. A total of 55 patients met the criteria of recurrent VAP. Of these 55 recurrences, 19 (35%) were relapses. Acute Physiology and Chronic Health Evaluation II score, injury severity score, and GCS were not associated with VAP relapse by univariate analyses. Patients who relapsed had primary VAP involving nonfermenting gram-negative bacilli (NFGNB) (Acinetobacter, Pseudomonas, and Stenotrophomonas species) more frequently than other organisms (68% vs. 32%, p = 0.001). Primary VAP with NFGNB was found to be a significant predictor of VAP relapse by univariate and multivariate logistic regression analysis (OR = 5.1, p = 0.003; OR = 4.63, p = 0.005, respectively). There is a high rate of VAP relapse associated with primary infection by NFGNB, suggesting initial treatment failure. Trauma patients with primary VAP involving these organisms may benefit from increased surveillance for relapse.

Simultaneous Degradation of Organophosphates and 4-Substituted Phenols by Stenotrophomonas Species LZ-1 with Surface-Displayed Organophosphorus Hydrolase

Organophosphorous hydrolase (OPH) was expressed onto the surface of a Stenotrophomonas species (LZ-1), capable of simultaneously degrading 4-substituted phenols, using the N- and C-terminal domains of ice nucleation protein (INPNC) as an anchoring motif for the first time. The engineered strain LZ-1 could degrade p-nitrophenyl-substituted organophosphates as well as their hydrolytic product, PNP, rapidly. Especially, addition of 4-CP (below 0.8 mM) significantly accelerated the complete degradation of above organophosphates (47.1, 34.0, and 40% reduction of time of paraoxon, parathion, and methyl-parathion, respectively) through the accelerated degradation of PNP due to enhanced cell growth supported by 4-CP as the carbon source. OPH could be surface-displayed at a high level without inhibition of cell growth and OPH activity in the presence of 4-CP. In soil samples, strain LZ-1 could also remove these compounds successfully. Functional display of heterologous proteins on the surface of indigenous bacteria could provide a promising technology for effective bioremediation of sites contaminated with mixed organic pollutants.

Bacterial contamination of cystic fibrosis clinics

Background Respiratory pathogens from CF patients can contaminate inpatient settings, which may be associated with increased risk of patient-to-patient transmission. Few data are available that assess the rate of bacterial contamination of outpatient settings. We determined the frequency of contamination of CF clinics and the effectiveness of alcohol-based disinfectants in reducing hand carriage of bacterial pathogens. Methods We conducted a point prevalence survey and before–after trial in outpatient clinics at 7 CF centers. The study examined CF patients with positive respiratory cultures for Pseudomonas, Staphylococcus, Stenotrophomonas or Burkholderia species. Hand carriage and environmental contamination with respiratory pathogens were assessed during clinic visits (Part I) and the effectiveness of hand hygiene performed by CF patients (Part II) was determined using molecular typing of recovered isolates. Results In Part I ( n = 97), the contamination rate was 13.6%. Pseudomonas and S. aureus, including methicillin-resistant strains, were cultured from patients' hands (7%), the exam room air (8%), and less commonly, environmental surfaces (1%). In Part II ( n = 100), the hand carriage rate of pathogens was 13.5% and 4 participants without initial detection of pathogens had hand contamination when recultured at the end of the clinic visit. Conclusions Respiratory pathogens from CF patients can contaminate their hands and the clinic environment, but the actual risk of patient-to-patient transmission in the outpatient setting remains difficult to quantify. These findings support several recommendations CF infection control recommendations including hand hygiene for staff and patients, contact precautions for certain pathogens, and disinfecting equipment and surfaces touched by patients and staff.

Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov., two nitrate-reducing bacteria isolated from soil

Three Gram-negative, rod-shaped, non-spore-forming, nitrate-reducing isolates (R-32746, R-32768(T) and R-32729(T)) were obtained from soil. Analysis of repetitive sequence-based PCR showed that the three isolates represented two different strains. 16S rRNA gene sequence analysis and DNA-DNA hybridization placed them within the genus Stenotrophomonas and revealed that they were genotypically different from each other and from all recognized Stenotrophomonas species. Analysis of the fatty acid composition and physiological and biochemical tests allowed differentiation from their closest phylogenetic neighbours. They are therefore considered to represent two novel species, for which the names Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov. are proposed, with strains R-32768(T) (=LMG 23958(T)=DSM 18941(T)) and R-32729(T) (=LMG 23959(T)=DSM 18929(T)), respectively, as the type strains.

Ventilator-Associated Pneumonia in the Home Care Setting

To describe the rate of infection, associated organisms, and potential risk factors for ventilator-associated pneumonia (VAP) in patients receiving mechanical ventilation at home. Retrospective cohort study. University-affiliated home care service. Patients receiving mechanical ventilation at home from June 1995 through December 2001. Fifty-seven patients underwent ventilation at home for a total of 50,762 ventilator-days (mean +/- SD, 890.6 +/- 644.43 days; range, 76-2,458 days). Seventy-nine episodes of VAP occurred in 27 patients (rate, 1.55 episodes per 1,000 ventilator-days). The first episode of VAP occurred after a mean (+/-SD) of 245 +/- 318.07 ventilator-days. VAP was most common during the first 500 days of ventilation. Rates of VAP were higher among patients who required ventilation for longer daily durations, compared with those who required it for shorter daily durations. There was no association of VAP with age, sex, underlying disease, reason for ventilation, antacid therapy, or steroid use. Microorganisms isolated from 33 episodes of VAP with available culture results included Pseudomonas species (17 isolates), Staphylococcus aureus (11), Serratia species (7), and Stenotrophomonas species (5). Eight patients died during the study; no deaths were attributed to pneumonia. Although the organisms associated with VAP in the home setting are similar to those associated with hospital-acquired VAP, the incidence and mortality is much lower in the home care setting. Interventions to reduce the risk of VAP among patients receiving home care should be focused on patients who require ventilation for longer daily durations or who are new to receiving mechanical ventilation at home.

Taxonomic diversity of predominant culturable bacteria associated with microaggregates from two different agroecosystems and their ability to aggregate soil in vitro

Soil macroaggregates (>250 μm) are easily disrupted when wetted quickly, whereas microaggregates (50–250 μm) are highly stable. There is little knowledge of functional groups of bacteria from microaggregates that can stabilize soil and their diversity. We isolated the predominant bacteria from microaggregates collected from two agroecosystems: a site cropped to barley (40 years with tillage) and to alfalfa (9 years with no tillage) and assayed for their potential to aggregate soil in an in vitro assay. Soil suspensions from physically disrupted microaggregates were spiral-plated and bacterial colonies were isolated from the most dilute portion of the spiral then purified. To characterize the culturable bacteria thus isolated, fatty acid methyl ester (FAME) profiles, substrate utilization patterns (Biolog), and DNA sequence analysis using 16s rRNA amplification region were performed for bacterial identification. Principal component analysis of FAME profiles and Biolog patterns was conducted to describe patterns of community composition of bacterial species within each agroecosystem. Gram-negative bacteria in microaggregates from barley soil consisted entirely of Pseudomonas spp. whereas Stenotrophomonas, Sphingobacterium, Chryseobacterium, and Acinetobacter spp. were found in addition to the Pseudomonas in microaggregates of alfalfa soil. Artificial aggregates were generated by amending a sandy soil with pure cultures of various species identified from microaggregates of both soils and tested for their water stability and their strength. The results indicated that Stenotrophomonas and Sphingobacterium species exhibited the greatest ability to stabilize soil and to increase aggregate strength among several species tested. This study demonstrated that microaggregates harbor functional groups of bacterial species capable of aggregating soil particles in vitro, and there were a greater range of such species in microaggregates of non-disturbed soil (alfalfa) than in disturbed, yearly tilled soil (barley).

Stenotrophomonas dokdonensis sp. nov., isolated from soil

A Gram-negative, rod-shaped, Stenotrophomonas-like bacterial strain, DS-16(T), was isolated from soil from Dokdo, Korea, and subjected to a polyphasic taxonomic study. Strain DS-16(T) grew optimally at pH 6.0-7.0 and 30 degrees C in the presence of 0.5 % (w/v) NaCl. It contained Q-8 as the predominant ubiquinone and iso-C(16 : 0), iso-C(15 : 0) and iso-C(17 : 1)omega9c as the major fatty acids. The DNA G + C content was 65.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DS-16(T) joined the cluster comprising Stenotrophomonas species. The levels of 16S rRNA gene sequence similarity between strain DS-16(T) and the type strains of Stenotrophomonas species ranged from 95.5 to 97.5 %. DNA-DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness of strain DS-16(T), demonstrated that this novel strain differs from Stenotrophomonas species with validly published names. On the basis of phenotypic, phylogenetic and genetic data, strain DS-16(T) (=KCTC 12543(T) = CIP 108839(T)) should be classified in the genus Stenotrophomonas as a member of a novel species, for which the name Stenotrophomonas dokdonensis sp. nov. is proposed.

Stenotrophomonas koreensis sp. nov., isolated from compost in South Korea

A Gram-negative, rod-shaped, non-spore-forming bacterium (TR6-01T) was isolated from compost near Daejeon city in South Korea. On the basis of 16S rRNA gene sequence similarity, strain TR6-01T was shown to belong to the class Gammaproteobacteria, related to Stenotrophomonas acidaminiphila (97.1 %) and Stenotrophomonas maltophilia (96.9 %); the phylogenetic distance from any other established species within the genus Stenotrophomonas was less than 97.0 %. Phenotypic and chemotaxonomic data (major ubiquinone Q-8; fatty acid profile) supported the affiliation of strain TR6-01T to the genus Stenotrophomonas. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain TR6-01T from the five Stenotrophomonas species with validly published names. TR6-01T therefore represents a novel species, for which the name Stenotrophomonas koreensis sp. nov. is proposed, with the type strain TR6-01T (= KCTC 12211T = JCM 13256T).

Nature of bacteria found on some wards in Sultan Qaboos University Hospital, Oman

This study aims to determine what objects lying in the hospital environment or brought in from outside contribute to the introduction of bacteria associated with nosocomial infections. One hundred swab specimens collected from children’s toys, sinks, door handles, telephone handsets and flowers brought into the hospital were plated on different culture media. Colonial growth on the media was purified and identified subsequently using standard bacteriological methods. Of the 100 samples cultured, 61 (61%) grew a range of bacteria including Pseudomonas aeruginosa (n=14, 23.0%), Acinetobacter spp. (n=13, 21.3%), Serratia spp. (n=9, 14.7%), Staphylococcus epidermidis (n=9, 14.7%), Stenotrophomonas maltophilia (n=4, 6.6%), Staphylococcus aureus (n=4, 6.6%), Enterobacter cloacae (n=3, 4.9%), Pantoea sp. (n=2, 3.3%), Chryseobacterium sp. (n=2, 3.3%) and Klebsiella pneumoniae (n=1, 1.6%). Although all the Serratia, Enterobacter, Klebsiella and Pantoea species isolates showed varying degrees of resistance to gentamicin, ceftriaxone, cefuroxime and cefotaxime, all were resistant to ampicillin. Chryseobacterium and Stenotrophomonas species isolates were resistant to amikacin, imipenem, gentamicin and ceftazidime, to which only three isolates of Pseudomonas species were resistant. All the staphylococcal isolates were susceptible to methicillin. Although there has been no major outbreak of a nosocomial infection in the hospital, it is strongly recommended that effective control measures (e.g., sampling the hospital water supply, disinfecting children’s toys, use of appropriate hand washing and checking some of the disinfectants for presence of bacteria) are needed. These measures are necessary to ensure that the antibiotic-resistant strains identified in this study are not allowed to spread in the hospital.

Synthesis of the compatible solutes glucosylglycerol and trehalose by salt-stressed cells of Stenotrophomonas strains

In this study, physiological processes were analysed, which are involved in salt acclimation of two Stenotrophomonas species, Stenotrophomonas maltophilia strain DSM 50170 and Stenotrophomonas rhizophila strain DSM 14405. S. maltophilia accumulated trehalose as the only osmolyte, whereas S. rhizophila produced additionally to trehalose glucosylglycerol (GG). The different spectrum and amounts of compatible solutes in these two strains led to differences in terms of their salt tolerance. The human-associated S. maltophilia was able to grow in media containing up to 3% NaCl (w/v). In contrast, S. rhizophila propagated in salinities up to 5% NaCl (w/v). The strain was isolated from the rhizosphere, a microenvironment which is characterised by high and changing salinities. Light microscopic analysis of S. rhizophila cells showed a significant increase in cell length of salt-treated cells in comparison to control cells. Cells of S. rhizophila exposed to more than 2% NaCl excreted GG into the medium during the transition from exponential to stationary growth phase, while the internal trehalose pool remained constant. This feature offers a high potential for the biotechnological production of GG.

Pseudoxanthomonas mexicana sp. nov. and Pseudoxanthomonas japonensis sp. nov., isolated from diverse environments, and emended descriptions of the genus Pseudoxanthomonas Finkmann et al. 2000 and of its type species

Three mesophilic bacteria (strains AMX 26B(T), UR374_02 and 12-3(T)) isolated respectively from an anaerobic digester, human urine and urban riverside soil were characterized. Cells were Gram-negative, motile, non-sporulating, straight to curved rods with one polar flagellum and had a strictly respiratory metabolism with O(2) as the preferential terminal electron acceptor. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all strains clustered within the Xanthomonadaceae branch of the Proteobacteria. Isolates AMX 26B(T) and UR374_02 exhibited 100 % 16S rRNA gene sequence similarity and both were related to strain 12-3(T) (99.6 % similarity). The closest relative of all the isolates was Pseudoxanthomonas broegbernensis DSM 12573(T) (similarity 97.1-97.5 %), and they were equidistantly related to Xanthomonas species (95.4-96.6 %), Stenotrophomonas species (95.3-96.1 %) and Pseudoxanthomonas taiwanensis ATCC BAA-4040(T) (95.3-95.4 %). Chemotaxonomic and biochemical data (branched-chain cellular fatty acid pattern without C(13 : 0) iso 3-OH, ubiquinone with eight isoprenoid units, limited range of substrates used, ability to reduce nitrite but not nitrate with the production of N(2)O) supported their affiliation to the genus Pseudoxanthomonas. The results of DNA-DNA hybridization and/or phenotypic analysis allowed them to be differentiated from the two Pseudoxanthomonas species with validly published names and showed that strain 12-3(T) was genomically and phenotypically distinct from the other two isolates. On the basis of these results, two novel species of the genus Pseudoxanthomonas are proposed: Pseudoxanthomonas mexicana sp. nov., consisting of strains AMX 26B(T) (=ATCC 700993(T)=CIP 106674(T)=JCM 11524(T)) (type strain) and UR374_02 (=DSM 15133), and Pseudoxanthomonas japonensis sp. nov., consisting of strain 12-3(T) (=CCUG 48231(T)=CIP 107388(T)=JCM 11525(T)). The report of these two novel species leads to the emendation of the description of the genus Pseudoxanthomonas and the re-evaluation of the phenotype of P. broegbernensis DSM 12573(T) necessitates the emendation of its description.

Central venous catheter-related bacteremia due to gram-negative bacilli: significance of catheter removal in preventing relapse.

To study the characteristics of catheter-related, gram-negative bacteremia (GNB) and the role of central venous catheter (CVC) removal. This retrospective study involved a search of the microbiological department records of CVC and blood cultures and patients' medical records. University of Texas M. D. Anderson Cancer Center, a tertiary-care hospital in Houston, Texas. Patients with cancer who had catheter-related GNB, defined as (1) a positive catheter tip culture with at least 15 colony-forming units semiquantitatively, (2) isolation of the same organism from the tip and peripheral blood cultures, (3) no other source for bacteremia except the CVC, and (4) clinical manifestations of infection (fever or chills). Between January 1990 and December 1996, 72 cases of catheter-related GNB were available for review. Most of the patients (67; 93%) had their CVCs removed in response to the bacteremia. Few patients (5; 7%) retained their CVCs and were treated with appropriate antibiotics. When CVCs were removed, only 1 patient (1%) relapsed with the same organism, whereas all 5 patients with retained CVCs relapsed after having responded (P < .001). The most commonly isolated organisms were Enterobacter, Klebsiella, Stenotrophomonas, Pseudomonas, and Acinetobacter species. Catheter removal within 72 hours of the onset of the catheter-related GNB was the only independent protective factor against relapse of the infection (odds ratio, 0.13; 95% confidence interval, 0.02-0.75; P = .02). In patients with documented catheter-related GNB, CVCs should be removed within 48 to 72 hours to prevent relapse.

Stenotrophomonas africana Drancourt et al. 1997 is a later synonym of Stenotrophomonas maltophilia (Hugh 1981) Palleroni and Bradbury 1993.

Type and reference strains of Stenotrophomonas maltophilia and Stenotrophomonas africana were compared with each other and with the type strains of other Stenotrophomonas species, using SDS-PAGE of whole-cell proteins, DNA-DNA hybridization and extensive biochemical characterization. S. maltophilia LMG 958T and S. africana LMG 22072T had very similar whole-cell-protein patterns and were also biochemically very similar. A DNA-DNA binding level of 70% between both type strains confirmed that S. africana and S. maltophilia represent the same taxon. It is concluded that S. africana is a later synonym of S. maltophilia.

Increase in methicillin-resistant Staphylococcus aureus acquisition and change in pathogen pattern associated with outbreaks of severe acute respiratory syndrome (SARS)

The pattern of endemic pathogenic isolates and the rates of acquisition of methicillin-resistant Staphylococcus aureus (MRSA) were evaluated in our 22-bed ICU during a SARS outbreak. This period was characterized by a change in patient case-mix, high use of steroids, change in the pattern of antibiotic prescribing, upgrading of infection control precautions, and wearing gloves all the time. These changes were associated with an increase in the isolation of MRSA, Candida and Stenotrophomonas species. On the other hand, Pseudomonas and Klebsiella species, which used to be endemic, had disappeared. The MRSA ICU acquisition rate was significantly increased during SARS (25.3% vs 5.0% in 2000, 3.8% in 2001, 2.9% in 2002, P < 0.001). This showed that changes within an ICU during a SARS outbreak could lead to a change in the pathogen pattern, and a significant increase in MRSA acquisition. Table 1

Effects of Empiric Antibiotic Administration for Suspected Pneumonia on Subsequent Opportunistic Pulmonary Infections

Optimal guidelines for empiric antibiotic (EAB) therapy in cases of suspected post-traumatic ventilator-associated pneumonia (VAP) are not well defined. EAB administration is thought to increase the incidence of opportunistic organisms; however, culture-directed (as opposed to empiric) treatment may delay antibiotic administration with possible adverse consequences. Our goal was to examine the impact of EAB administration on the incidence of subsequent VAP and opportunistic organisms in a series of critically injured patients with sepsis syndrome. This is a retrospective review of all patients admitted to a Level I trauma center who underwent multiple fiberoptic bronchoscopies (FOBs) for diagnosis of suspected VAP as the cause of postinjury sepsis syndrome. The relationships between EAB administration, positive cultures (>10(5) colony-forming units) at repeat FOB, and prevalence of opportunistic organisms (methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas species, Acinetobacter species, and/or yeast) were evaluated. Over a 13-month period ending on January 1, 1999, 36 intubated trauma patients underwent more than one FOB during their intensive care unit stay. Twenty-nine patients received EAB immediately after the initial FOB. There was no difference in the rate of EAB administration in patients who developed subsequent VAP after initial FOB (19 of 24, 79%) versus those who did not develop VAP (10 of 12, 83%; P = 0.65). There were 30 VAPs diagnosed in 58 subsequent FOBs (i.e., after the initial FOB) of which 23 were due to an opportunistic organism compared with two VAPs due to an opportunistic organism diagnosed at initial FOB (P < 0.001). Twenty-one of the 23 opportunistic VAPs at subsequent FOBs had received EAB before the first FOB compared with four of seven nonopportunistic organisms (P = 0.06). Administration of EAB does not impact the incidence of subsequent VAP. However, EAB may be related to the development of subsequent opportunistic pulmonary infections.

In vitro susceptibility to pexiganan of bacteria isolated from infected diabetic foot ulcers

During two clinical trials involving the treatment of 835 outpatients with infected diabetic foot ulcers, 2515 bacterial isolates, including 2337 aerobes and 178 anaerobes, were grown from cultures of the ulcers. The in vitro susceptibility of these isolates was determined to pexiganan, a peptide anti-infective evaluated in these clinical trials, and to other classes of antibiotics. Pexiganan demonstrated broad spectrum antimicrobial activity against Gram-positive and Gram-negative aerobes and anaerobes. The MIC 90 values for the most common species among 1735 Gram-positive aerobes isolated, such as Staphylococcus aureus, coagulase-negative staphylococci, Group A streptococci, and Group B streptococci , were 16 μg/mL or less. Of 602 Gram-negative aerobes tested, the MIC 90 values for pexiganan were 16 μg/mL or less for Acinetobacter, Pseudomonas, Stenotrophomonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, and Flavobacterium species. Pexiganan had a MIC 90 of 4 to 16 μg/mL against the anaerobic isolates of Bacteroides, Peptostreptococcus, Clostridium, and Prevotella species. Importantly, pexiganan did not exhibit cross-resistance with other commonly used antibiotics, including β-lactams, quinolones, macrolides, and lincosamides. The broad spectrum in vitro antimicrobial activity of pexiganan against clinical isolates from infected diabetic foot ulcers supports its potential as a local therapy for infected diabetic foot ulcers.

Purification and properties of extracellular carboxyl proteases of acid-tolerant bacteria, isolated from compost.

Four strains of acid-tolerant and protein-using bacteria were isolated from compost. Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively. Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium. The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55 degrees C. Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP. These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.