Assessment of the effect of hand dryers used in shopping malls on hand hygiene

BackgroundHand drying is one of the most important factors affect hand hygiene. This study was conducted to investigate the effect of hand dryers used in the restrooms located on the food court floors in shopping malls in Turkey on hand hygiene.MethodsHands were washed for at least 20 seconds by following hygienic hand washing procedures of the World Health Organization. Swab samples were taken from the wet hands after hand washing, then from the dry hands, which were dried under the hand dryers and from the air blowing part of the hand dryers. Samples were cultured on agar plates that were directly exposed to the air-blowing part of the hand dryers.ResultsIt was found that total coliform bacteria were 0.000 colony-forming unit (CFU)/petri in wet and dried hand, 3.437 CFU/petri in blown air and 5.250 CFU/petri in swab samples. Staphylococcus aureus was found to be 0.125, 64.125, 26.375, and 388.750 CFU/petri, respectively. Total bacteria count was found to be 0.687, 48.750, 35.625, and 595.000 CFU/petri, respectively. S. aureus and the total bacterial load were higher in the blower outlet of the hand dryers than washed hand, blown air and dried hand (P < .05). The bacteria count in the unfiltered hand dryers was higher than that in the filtered hand dryers (P < .05).ConclusionsUsing hand dryers would negatively affect hand hygiene even if hands were washed following hygienic hand washing procedures.     

Evaluation of phagocytic and bactericidal activities of neutrophil granulocytes. Determination of viable extracellular bacteria by their incorporation of 14C-leucine and 3H-thymidine.

A method for evaluation of human neutrophil granulocyte function based on the combined determination of total and extracellular bacteria is described. The total number of surviving bacteria is assessed by the determination of colony forming units (CFU) after hypotonic lysis of granulocytes. Extracellular viable bacteria can be determined by the incorporation of 14C-leucine or 3H-thymidine into bacterial macromolecules since there is a linear relationship between macromolecular synthesis and bacterial number and insignificant amounts of both 14C-leucine and 3H-thymidine is taken up by intracellular viable organisms. This method might be suitable for the differentiation of defects in phagocytosis and intracellular killing of various bacteria.     

A problem of hospital hygiene: the presence of aspergilli in hospital wards with different air-conditioning features

A total of 1,030 microbiological samples were taken in 3 hospital wards with different air-conditioning features: no conditioning system (ward A), a conditioning system equipped with minimum efficiency reporting value (MERV) filters (ward B), and a conditioning system thoroughly maintained and equipped with high-efficiency particulate air (HEPA) filters (absolute) (ward C). The air in each ward was sampled, and the bacterial and fungal concentrations were determined by active and passive methods. The concentration of fungi on surfaces was also determined. Active sampling showed positive samples in wards A and B only, with average values of 0.50 colony-forming units (CFU)/m(3) (95% CI, 0.30 to 0.70) in A and 0.16 CFU/m(3) (95% CI, 0.13 to 0.20) in B. Passive sampling was positive only in ward A (mean, 0.14 CFU/cm(2)/h; 95% CI, 0.13 to 0.15). Aspergillus was found in 27% and 22% of sampled surfaces in wards A and B, respectively, but in no samples from ward C. The most commonly found species was A. fumigatus (76% of cases in A and 34% of cases in B). The results show that the use of air-conditioning systems markedly reduces the concentration of aspergilli in the environment. Proper maintenance of these systems is clearly fundamental if their efficacy is to be ensured.     

No relationship between gastric pH, small bowel bacterial colonisation, and diarrhoea in HIV-1 infected patients

Background/Aims—Conclusivestudies of small bowel bacterial overgrowth in patients with HIV-1infection are limited. The relation was therefore determined betweenthe quantity and species of bacteria in the proximal small intestine ofHIV-1 infected patients and the presence of diarrhoea, gastric acidity,severity of immune deficiency, and clinical outcome.
Methods—Bacteria inthe duodenal fluids obtained endoscopically from 32 HIV-1 infectedpatients, 21 of whom had diarrhoea, and seven control subjects withoutHIV-1 risk factors were quantified and speciated. Gastric pH wasdetermined at the time of endoscopy. Clinical follow up was performedto assess outcome.
Results—OropharyngealGram positive cocci were present in fluids from 28 patients (88%).Gram negative aerobic or facultatively anaerobic bacteria were presentin fluids from 12 patients (38%), and strict anaerobes were detectedin six patients (19%), but for both groups colony counts infrequentlyexceeded 10⁴ colony forming units/ml. The number andspecies of bacteria did not correlate with the presence of diarrhoea,gastric pH, or CD4 lymphocyte count.
Conclusions—Smallbowel bacterial overgrowth is not common in HIV-1 infected patients,regardless of the presence of diarrhoea, and is not associated with hypochlorhydria.

Keywords:bacterial overgrowth; diarrhoea; HIV infection; AIDS

     

Defining lower airway bacterial infection in children with chronic endobronchial disorders

Background

Differentiating lower airway bacterial infection from possible upper airway contamination in children with endobronchial disorders undergoing bronchoalveolar lavage (BAL) is important for guiding management. A diagnostic bacterial load threshold based on inflammatory markers has been determined to differentiate infection from upper airway contamination in infants with cystic fibrosis, but not for children with protracted bacterial bronchitis (PBB), chronic suppurative lung disease (CSLD), or bronchiectasis.

Methods

BAL samples from children undergoing bronchoscopy underwent quantitative bacterial culture, cytologic examination, and respiratory virus testing; a subset also had interleukin‐8 examined. Geometric means (GMs) of total cell counts (TCCs) and neutrophil counts were plotted by respiratory pathogen bacterial load. Logistic regression determined associations between age, sex, Indigenous status, antibiotic exposure, virus detection and bacterial load, and elevated TCCs (>400 × 10³ cells/mL) and airway neutrophilia (neutrophils >15% BAL leukocytes).

Results

From 2007 to 2016, 655 children with PBB, CSLD, or bronchiectasis were enrolled. In univariate analyses, Indigenous status and bacterial load ≥10⁵ colony‐forming units (CFU)/mL were positively associated with high TCCs. Viruses and bacterial load ≥10⁴ CFU/mL were positively associated with neutrophilia; negative associations were seen for Indigenous status and macrolides. In children who had not received macrolide antibiotics, bacterial load was positively associated in multivariable analyses with high TCCs at ≥10⁴ CFU/mL and with neutrophilia at ≥10⁵ CFU/mL; GMs of TCCs and neutrophil counts were significantly elevated at 10⁴ and 10⁵ CFU/mL compared to negative cultures.

Conclusions

Our findings support a BAL threshold ≥10⁴ CFU/mL to define lower airway infection in children with chronic endobronchial disorders.

     

The probiotic Lactobacillus fermentum 296 attenuates cardiometabolic disorders in high fat diet-treated rats

Background and aimHigh-fat (HF) diet consumption has been associated with gut dysbiosis and increased risk of dyslipidemia, type 2 diabetes mellitus and hypertension. Probiotic administration has been suggested as a safe therapeutic strategy for the treatment of cardiometabolic disorders. This study was designed to assess the effects of probiotic Lactobacillus (L.) fermentum 296, a fruit-derived bacteria strain, against cardiometabolic disorders induced by HF diet.Methods and resultsMale Wistar rats were divided into control diet (CTL); HF diet; and HF diet treated with Lactobacillus fermentum 296 (HF + Lf 296). The L. fermentum 296 strain at 1 × 10⁹ colony forming units (CFU)/ml were daily administered by oral gavage for 4 weeks. The results showed that rats fed with HF diet displayed insulin resistance, reduced Lactobacillus spp. counts in feces, serum lipids, and oxidative profile. Rats fed on HF diet also demonstrated augmented blood pressure associated with sympathetic hyperactivity and impaired baroreflex control. The administration of L. fermentum 296 for 4 weeks recovered fecal Lactobacillus sp. counts and alleviated hyperlipidemia, sympathetic hyperactivity, and reduced systolic blood pressure in HF rats without affecting baroreflex sensibility.ConclusionOur results suggest the ability of L. fermentum 296 improve biochemical and cardiovascular parameters altered in cardiometabolic disorders.     

Low-grade small intestinal bacterial overgrowth is common in patients with non-alcoholic steatohepatitis on quantitative jejunal aspirate culture

Background

Though pathogenesis of non-alcoholic steatohepatitis (NASH) is unclear, association with small intestinal bacterial overgrowth [SIBO] and fecal dysbiosis is suggested. We evaluated SIBO in NASH using quantitative jejunal aspirate culture (conventional criteria: ≥?10⁵ colony forming unit (CFU)/mL and newer cutoff ≥?10³ CFU/mL) and glucose hydrogen breath test.

Methods

Thirty-eight patients with NASH (age 37.5 years, range 20–54, 9, 24% female), diagnosed by ultrasonography, alanine aminotransferase >1.5 times normal and liver biopsy (in 27/38, 71%) and exclusion of other causes and 12 constipation-predominant irritable bowel syndrome as historical controls (age 39.5-y, 26–44; 3, 25% female) without fatty liver were studied.

Results

Jejunal aspirates, obtained in 35/38 patients, were sterile in 14/35 (40%) and bacteria isolated in 21 (60%) (all aerobic, in one anaerobe also; Gram positive 5, negative 13, both 3). In contrast, bacteria (two Gram negative) were isolated in 3/12 (25%) controls (odds ratio 4.5, 95% CI 1.0–19.5; p = 0.04); colony counts were higher in NASH than controls (median 380 CFU/mL, 0–200,000 vs. 0 CFU/mL, 0–1000; p = 0.02). Gram negative bacteria tended to be commoner in NASH than controls (16/35 vs. 2/12; p = 0.07). Seven out of 35 (20%) patients with NASH (≥?10⁵ CFU/mL in 5 and 2 other on glucose hydrogen breath test) and no control had SIBO (p = ns); low-grade SIBO (≥10³ CFU/mL) was commoner in NASH than controls (14/35, 40%, vs. 1/12, 8.3%; p = 0.04). There was no correlation between bacterial colony count and bacterial type and anthropometric and biochemical parameters.

Conclusion

Low-grade bacterial overgrowth, particularly with Gram negative bacteria, was commoner in NASH than controls.

     

Detection of intestinal colonization of probiotic Lactobacillus rhamnosus by stool culture in modified selective media

ObjectiveTo develop an approach for making a selective and differential media specific for Lactobacillus rhamnosus Goldin and Gorbach (LGG) in order to distinguish it from other bacterial flora in fecal samples.MethodsThe current media referred as LGGM has been prepared by using MRS media ingredients (replacing dextrose with 2% sorbitol), and incorporation of nalidixic acid (40 μg/mL), bromocresol purple (0.002 %); the pH of the media was adjusted to 4.5.ResultsThe LGGM showed a greater colony forming units compared to MRS media. The growth of pure LGG strain was significantly greater in LGGM, and the recovery rate of LGG released from fecal samples (after oral feeding) was significantly greater (P<0.001).ConclusionsThus LGGM could be used as a potential media for the isolation and identification of LGG strain.     

In vitro antiplasmodial activity of marine sponge Stylissa carteri associated bacteria against Plasmodium falciparum

ObjectiveTo identify the possible antiplasmodial drugs from bacteria associated with marine sponge Stylissa carteri (S. carteri).MethodsThe S. carteri samples were collected from Thondi coast and subjected for enumeration and isolation of associated bacteria. Filter sterilized extracts (100, 50, 25, 12.5, 6.25 and 3.125 μ g/mL) from isolated bacterial strains were screened for antiplasmodial activity against Plasmodium falciparum (P. falciparum) and potential extracts were also screened for biochemical constituents.ResultsTwelve samples of S. carteri were collected and subjected for enumeration and isolation of associated bacteria. The count of bacterial strains were maximum in November 2007 (34 × 10⁴ CFU/g) and the average count was maximum during the monsoon season (203 × 10³ CFU/g). Thirty two morphologically different bacterial strains were isolated from S. carteri and the ethyl acetate bacterial extracts were screened for antiplasmodial activity against P. falciparum. The antiplasmodial activity of a strain THB17 (IC₅₀ 20.56 μ g/mL) extract is highly comparable with the positive control chloroquine (IC₅₀ 19.59 μ g/mL) and 13 bacterial extracts which showed IC₅₀ value of more than 100 μ g/mL. Statistical analysis reveals that, significant in vitro antiplasmodial activity (P<0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial strains after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of reducing sugars and alkaloids in the ethyl acetate extracts of bacterial strains.ConclusionsThe ethyl acetate extract of THB17 possesses lead compounds for the development of antiplasmodial drugs.     

In vitro antiplasmodial activity of Clathria vulpina sponge associated bacteria against Plasmodium falciparum

ObjectiveTo identify the possible antiplasmodial drugs from bacteria associated with marine sponge Clathria vulpina (C. vulpina).MethodsThe C. vulpina samples were collected from Thondi coast and subjected to enumeration and isolation of associated bacteria. Filtered and sterilized extracts (100, 50, 25, 12.5, 6.25 and 3.125 μg/mL) from isolated bacterial strains were screened for antiplasmodial activity against Plasmodium falciparum. Potential extracts were also screened for biochemical constituents.ResultsThirty one bacterial strains were isolated from twelve sponge samples collected from Thondi coast and screened for antiplasmodial assay. The count of bacterial strains were maximum in November 2007 (19×10⁴ CFU/g) and the average count was maximum during the monsoon season (110×10³ CFU/g). The antiplasmodial activity of strain THB15 was highly comparable (IC₅₀ = 20.73 μg/mL) with the positive control chloroquine (IC₅₀ = 19.59 μg/mL) and 21 bacterial strains showed IC₅₀ value of more than 100 μg/mL. Statistical analysis reveals that, significant in vitro antiplasmodial activity (P<0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial strains after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of sugars and alkaloids in the ethyl acetate extracts of bacterial strains.ConclusionThe ethyl acetate extract of THB15 possesses lead compounds for the development of antiplasmodial drugs.     

Biocide activity of microfiber mops with and without silver after contamination

ObjectiveThe purpose of the present research was to compare the residual microbial load in Solo System microfiber mops with silver and in normal microfiber mops without silver to see whether those with the silver prevent bacterial proliferation and spread more effectively during normal cleaning operations.MethodsMops with and without silver were experimentally contaminated with suspension of Staphylococcus aureus ATCC 6538. The bioburden was evaluated by a filtering procedure according to UNI EN 1174 after contamination, after washing and after different times of impregnation in an alcohol-base detergent.Results and discussionThe results obtained lead to the conclusion that silver microfiber mop was significantly more effective in reducing bacterial load despite initial high level contamination (10⁶-10⁷ CFU/50 cm²). Indeed, after low temperature washing, the bacterial load was already completely eliminated while the mop without silver still presented relatively high levels of the microorganism (approximately 10² CFU/50 cm²) even after being soaked for 8 hours in a detergent/disinfectant.     

Microbiological survey of dialysate: vantage of use of sterile bag concentrate

Haemodialysed patients are exposed to nearly 400 litres of dialysis water weekly. The bacterial contamination of treated dialysate and water induces acute pyrogenic reactions or chronic damage and cytokine activation. The aim of this study was to value the microbiological parameters of dialysis water and dialysate of our monitors by bacterial culture (measured as colony forming units [CFU]) of water samples at 37 degrees C after 48 hours, at 22 degrees C after 72 hours and after seven days, and by measuring endotoxin levels (endotoxin units [EU]). In our centre, there are 16 monitors (6 monitors use sterile dialysate fluid and 10 monitors use non sterile dialysate fluid). The chemicals used for disinfection are chlorine and paracetic acid. Water samples were taken under sterile procedures every three months for a year. No bacteria were found in the samples of water of the dialysis ring; EU were lower than the limit value of 0.25 EU/ml fixed by the European Pharmacopoeia. The concentration of CFU and EU of the dialysate, taken from monitors with a sterile bag, were lower than those of other monitors (p < 0.05 t Student test). However, the levels of CFU/ml and EU/ml of dialysate samples, taken from monitors with a non-sterile bag, were lower than the guideline value of the European Pharmacopoeia (v.n. CFU < 50 CFU/ml and EU < 0.05 EU/ml). Frequent examination of CFU and EU is essential to reduce the damage caused by the use of contaminated water, therefore the goal of future dialytic techniques will be the use of "sterile dialysate".     

Solubilización in vitro del aceite de árbol de té y primeros resultados de su efecto antifúngico en onicomicosis

IntroductionOnychomycosis is the main cause of nail alteration. Hepatotoxicity, interference and low adherence to pharmacological treatment are associated. Therefore, our objective was to assess the in vitro effectiveness of tea tree essential oil (less harmful) against main causative agents of these infections.Material and methodsTrichophyton rubrum and Trichophyton mentagrophytes were isolated and inoculated at a concentration of 3x10⁵ CFU / mL in potato agar dextrose and tea tree essential oil at different concentrations to assess its effect by counting colony forming units and radial growth.ResultsTrichophyton rubrum growth inhibition was obtained at concentrations higher than 0.04% of the essential tea tree oil (p = 0.004). In the case of Trichophyton mentagrophytes, inhibition was obtained at 0.02% (p = 0.017), and even complete inhibition at a final concentration of the oil at 0.07%.ConclusionsTea tree essential oil inhibits the in vitro growth of the fungus and may be a less harmful alternative to the onychomycosis treatment.     

An increase in non-toxigenic Corynebacterium diphtheriae infections in Poland — molecular epidemiology and antimicrobial susceptibility of strains isolated from past outbreaks and those currently circulating in Poland

ObjectivesAn increase in non-toxigenic Corynebacterium diphtheriae infections – mainly invasive infections – has been observed in countries with high vaccination coverage. However, reasons for this situation are unknown. In this study we characterized and compared human clinical isolates of non-toxigenic C. diphtheriae strains isolated from infections that have occurred over recent years and C. diphtheriae strains isolated from diphtheria cases from past outbreaks in Poland.MethodsWe determined biotypes, genotypes, the occurrence of plasmids, and antimicrobial susceptibilities of 19 clinical C. diphtheriae strains. Genotypes were determined using pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) techniques.ResultsThe non-toxigenic C. diphtheriae strains isolated over the last few years were found to belong to biotype gravis and were genetically indistinguishable using PFGE and ERIC-PCR techniques. No plasmids were detected in the strains. All tested strains were susceptible to penicillin and erythromycin, as well as to imipenem, vancomycin, daptomycin, gentamicin, tetracycline, clindamycin, trimethoprim/sulfamethoxazole, rifampin, quinupristin/dalfopristin, and linezolid. Of the strains tested, 47% were intermediate for cefotaxime.ConclusionsThe genetic similarity of non-toxigenic C. diphtheriae strains causing infection suggests that the strains represent a single clone. They may possess additional virulence genes in a chromosome, related with higher pathogenicity and invasiveness. The genetic changes have not been followed by resistance to antibiotics.     

Periodontitis increases risk of viable bacteria in freshly drawn blood donations

BackgroundThe aim of the study was to determine if periodontitis, which often causes transient bacteraemia, associates with viable bacteria in standard blood donations.Materials and methodsThis was a cross-sectional study of 60 self-reported medically healthy blood donors aged over 50 years. According to standard procedures, whole blood was separated by fractionation into plasma, buffy-coat, and red blood cell (RBC)-fractions. The buffy-coat was screened for bacterial contamination using BacT/ALERT. Samples from plasma and RBC-fractions were incubated anaerobically and aerobically at 37°C for 7 days on trypticase soy blood agar (TSA). For identification, colony polymerase chain reaction was performed using primers targeting 16S rDNA.ResultsFrom 62% of the donors with periodontitis, bacterial growth was observed on at least 1 out of 4 plates inoculated with plasma or RBCs, whereas only 13% of plates inoculated with plasma or RBCs from periodontally healthy controls yielded bacterial growth (relative risk 6.4, 95% CI: 2.1; 19.5; p=0.0011). None of the donors tested positive for bacterial contamination using BacT/ALERT. Cutibacterium acnes was found in 31% of the donations from donors with periodontitis and in 10% of the donations from periodontally healthy donors. In addition, Staphylococcus species, Bacillus mycoides, Aggregatibacter aphrophilus, and Corynebacterium kroppenstedtii were detected.DiscussionPeriodontitis increased the risk of bacterial contamination of blood products. Contaminating bacteria are often associated with the RBC-fraction. As the BacT/ALERT test is generally performed on platelet products, routine screening fails to detect many occurrences of viable bacteria in the RBC-fraction.     

Primary research on unrelated double umbilical cord blood transplantation and implantation kinetics

Abstract

Objective

This study sought to examine implantation and implantation kinetics in double umbilical cord blood transplantation (DUCBT).

Methods

Twenty-nine patients who underwent a two-unit unrelated donor cord blood transplantation were included in this study. After transplantation, hematopoietic chimerism of the peripheral blood was evaluated based on the results of short tandem repeat polymerase chain reaction. Using these results, we were able to judge whether the transplanted cells implanted, determine which donor's cells implanted, and further examine the kinetics of implantation in DUCBT. The numbers of total nucleated cells (TNCs), CD34⁺ cells, colony forming units (CFUs), colony forming unit-granulocytes and macrophages (CFU-GMs), and CD3⁺ cells were compared between the dominant units and the non-dominant units in an attempt to understand the discipline and implantation kinetics of DUCBT.

Results

Neither the TNC counts nor the counts of CD34⁺ cells, CFU, CFU-GM, or CD3⁺ cells were significantly different between the dominant units and the non-dominant units (P values of 0.584, 0.322, 0.842, 0.534, and 0.082, respectively).

Conclusions

We were able to determine the engraftment status at 14 days after DUCBT, although the implantation kinetics of DUCBT remain uncharacterized and require further research.     

Bacterial skin flora and contamination of blood components: do we defer blood donors wisely?

Background and objectives Bacterial infection through contaminated blood is currently the greatest infection risk in relation to a transfusion. Deferral of prospective blood donors with a skin disorder is a common practise, because bacteria usually originate from the donor’s skin. The effectiveness of current deferral guidelines to prevent the bacterial contamination of blood has not been assessed. Materials and methods We recruited 55 blood donors with a skin disorder that prevented donation, and matched three controls for each case. The donors filled out a questionnaire and one bacterial culture sample was taken from venepuncture forearm skin. Results The median total number of colony forming skin bacteria was significantly higher in the cases (224 CFUs per sample) than controls (105 CFU per sample). Staphylococcus aureus was significantly more often present on the skin in cases (49%) as compared to controls (7%). Regarding other bacterial genera, no difference between cases and controls was found. Conclusions This study shows that our current guidelines for deferral of blood donors with skin disorders effectively identifies individuals with a high number of bacteria on their skin, as well as S. aureus carriers. However, deferral due to skin disorders had only a minor impact on blood product contamination when compared to other actions.     

结核分枝杆菌低剂量感染小鼠模型的建立与分析

目的 建立结核分枝杆菌低剂量感染的小鼠模型,分析感染小鼠组织荷菌量、组织病理随感染时间的动态变化。 方法 以100 菌落形成单位(CFU)的结核分枝杆菌标准株H37Rv经尾静脉途径感染雌性C57BL/6J小鼠40只,于感染后1、3、5、8、12、16、20、24周取材,每个时间点5只小鼠,脾、肺组织匀浆培养检测脾脏组织的荷菌量,脾脏、肺脏及肝脏组织经HE染色分析病理变化。 结果 小鼠感染后3周脾脏荷菌量达到（4.97±0.19）lg CFU,在感染后5周下降至（3.64±0.22）lg CFU,至感染后8周降到最低的（2.75±0.23）lg CFU;感染后3周肝、脾、肺等脏器出现病理改变,感染5周时病变加重,感染8周时病变自然减轻。 结论 成功建立了结核分枝杆菌低剂量感染小鼠模型,为结核分枝杆菌慢性持续感染或潜伏感染的研究可提供有用的工具。     

High bacterial load in asymptomatic diabetic patients with neurotrophic ulcers retards wound healing after application of Dermagraft

Diabetic neuropathic foot ulcers are a major healthcare burden. These chronic wounds always have a bacterial load, and although normal flora is not harmful, increased tissue burden may impede healing before clinical signs of infection are evident. In this study, chronic noninfected diabetic neuropathic foot ulcers (those with adequate blood supply and pressure offloading) were assessed for bacterial burden immediately before the application of a skin substitute. Eight patients with diabetic neuropathic foot ulcers greater than 1 cm2 and free of necrotic tissue had 3-mm tissue biopsies taken from the ulcer base for quantitative bacteriology. Five of the eight patients (75%) had greater or equal to 10(5) colony forming units/gram organisms present despite the absence of clinical signs of infection. Wound healing rates were linked to bacterial load as determined from quantitative biopsy--no growth was associated with a wound healing rate of 0.2 cm per week, 10(5) to 10(6) colony forming units/gram was associated with a healing rate of 0.15 cm per week, and greater than 10(6) colony forming units/gram was associated with 0.05 cm/per week healing rate. High bacterial burden impeded healing both before and after the application of the skin substitute. The authors will change their clinical practice to assess all diabetic neuropathic foot ulcers using quantitative skin biopsies before applying skin substitutes. All patients will be treated with combination antibiotics and repeat biopsies obtained with decreased bacterial burden (< 10(6) colony forming units/gram) prior to using any bioengineered skin substitute or growth factor treatment.