The use of PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) to detect bacterial and fungal colonization in healthy military service members.

BACKGROUND: The role of microbial colonization in disease is complex. Novel molecular tools to detect colonization offer theoretical improvements over traditional methods. We evaluated PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) as a screening tool to study colonization of healthy military service members. METHODS: We assessed 101 healthy Soldiers using PCR/ESI-TOF-MS on nares, oropharynx, and groin specimens for the presence of gram-positive and gram-negative bacteria (GNB), fungi, and antibiotic resistance genes. A second set of swabs was processed by traditional culture, followed by identification using the BD Phoenix automated system; comparison between PCR/ESI-TOF-MS and culture was carried out only for GNB. RESULTS: Using PCR/ESI-TOF-MS, at least one colonizing organism was found on each individual: mean (SD) number of organisms per subject of 11.8(2.8). The mean number of organisms in the nares, groin and oropharynx was 3.8(1.3), 3.8(1.4) and 4.2(2), respectively. The most commonly detected organisms were aerobic gram-positive bacteria: primarily coagulase-negative Staphylococcus (101 subjects: 341 organisms), Streptococcus pneumoniae (54 subjects: 57 organisms), Staphylococcus aureus (58 subjects: 80 organisms) and Nocardia asteroides (45 subjects: 50 organisms). The mecA gene was found in 96 subjects. The most commonly found GNB was Haemophilus influenzae (20 subjects: 21 organisms) and the most common anaerobe was Propionibacterium acnes (59 subjects). Saccharomyces species (30 subjects) were the most common fungi detected. Only one GNB (nares E. coli) was identified in the same subject by both diagnostic systems. CONCLUSION: PCR/ESI-TOF-MS detected common colonizing organisms and identified more typically-virulent bacteria in asymptomatic, healthy adults. PCR/ESI-TOF-MS appears to be a useful method for detecting bacterial and fungal organisms, but further clinical correlation and validation studies are needed.

Staphylococcus aureus colonization of healthy military service members in the United States and Afghanistan.

BACKGROUND: Staphylococcus aureus [methicillin-resistant and methicillin-susceptible (MRSA/MSSA)] is a leading cause of infections in military personnel, but there are limited data regarding baseline colonization of individuals while deployed. We conducted a pilot study to screen non-deployed and deployed healthy military service members for MRSA/MSSA colonization at various anatomic sites and assessed isolates for molecular differences. METHODS: Colonization point-prevalence of 101 military personnel in the US and 100 in Afghanistan was determined by swabbing 7 anatomic sites. US-based individuals had received no antibiotics within 30 days, and Afghanistan-deployed personnel were taking doxycycline for malaria prophylaxis. Isolates underwent identification and testing for antimicrobial resistance, virulence factors, and pulsed-field type (PFT). RESULTS: 4 individuals in the US (4 isolates- 3 oropharynx, 1 perirectal) and 4 in Afghanistan (6 isolates- 2 oropharynx, 2 nare, 1 hand, 1 foot) were colonized with MRSA. Among US-based personnel, 3 had USA300 (1 PVL+) and 1 USA700. Among Afghanistan-based personnel, 1 had USA300 (PVL+), 1 USA800 and 2 USA1000. MSSA was present in 40 (71 isolates-25 oropharynx, 15 nare) of the US-based and 32 (65 isolates- 16 oropharynx, 24 nare) of the Afghanistan-based individuals. 56 (79%) US and 41(63%) Afghanistan-based individuals had MSSA isolates recovered from extra-nare sites. The most common MSSA PFTs were USA200 (9 isolates) in the US and USA800 (7 isolates) in Afghanistan. MRSA/MSSA isolates were susceptible to doxycycline in all but 3 personnel (1 US, 2 Afghanistan; all were MSSA isolates that carried tetM). CONCLUSION: MRSA and MSSA colonization of military personnel was not associated with deployment status or doxycycline exposure. Higher S. aureus oropharynx colonization rates were observed and may warrant changes in decolonization practices.

Multidrug-resistant gram-negative bacteria colonization of healthy US military personnel in the US and Afghanistan.

BACKGROUND: The US military has seen steady increases in multidrug-resistant (MDR) gram-negative bacteria (GNB) infections in casualties from Iraq and Afghanistan. This study evaluates the prevalence of MDR GNB colonization in US military personnel. METHODS: GNB colonization surveillance of healthy, asymptomatic military personnel (101 in the US and 100 in Afghanistan) was performed by swabbing 7 anatomical sites. US-based personnel had received no antibiotics within 30 days of specimen collection, and Afghanistan-based personnel were receiving doxycycline for malaria chemoprophylaxis at time of specimen collection. Isolates underwent genotypic and phenotypic characterization. RESULTS: The only colonizing MDR GNB recovered in both populations was Escherichia coli (p=0.01), which was seen in 2% of US-based personnel (all perirectal) and 11% of Afghanistan-based personnel (10 perirectal, 1 foot+groin). Individuals with higher off-base exposures in Afghanistan did not show a difference in overall GNB colonization or MDR E. coli colonization, compared with those with limited off-base exposures. CONCLUSION: Healthy US- and Afghanistan-based military personnel have community onset-MDR E. coli colonization, with Afghanistan-based personnel showing a 5.5-fold higher prevalence. The association of doxycycline prophylaxis or other exposures with antimicrobial resistance and increased rates of MDR E. coli colonization needs further evaluation.

Detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus colonization of healthy military personnel by traditional culture, PCR, and mass spectrometry.

BACKGROUND: Methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus colonization is associated with increased rates of infection. Rapid and reliable detection methods are needed to identify colonization of nares and extra-nare sites, particularly given recent reports of oropharynx-only colonization. Detection methods for MRSA/MSSA colonization include culture, PCR, and novel methods such as PCR/electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). METHODS: We evaluated 101 healthy military members for S. aureus colonization in the nares, oropharynx, axilla, and groin, using CHROMagar S. aureus medium and Xpert SA Nasal Complete PCR for MRSA/MSSA detection. The same subjects were screened in the nares, oropharynx, and groin using PCR/ESI-TOF-MS. RESULTS: By culture, 3 subjects were MRSA-colonized (all oropharynx) and 34 subjects were MSSA-colonized (all 4 sites). PCR detected oropharyngeal MRSA in 2 subjects, which correlated with culture findings. By PCR, 47 subjects were MSSA-colonized (all 4 sites); however, 43 axillary samples were invalid, 39 of which were associated with deodorant/anti-perspirant use (93%, p < 0.01). By PCR/ESI-TOF-MS, 4 subjects were MRSA-colonized, 2 in the nares and 2 in the oropharynx; however, neither of these correlated with positive MRSA cultures. Twenty-eight subjects had MSSA by PCR/ESI-TOF-MS, and 41 were found to have possible MRSA (S. aureus with mecA and coagulase-negative Staphylococcus (CoNS)). CONCLUSION: The overall 3% MRSA colonization rate is consistent with historical reports, but the oropharynx-only colonization supports more recent findings. In addition, the use of deodorant/anti-perspirant invalidated axillary PCR samples, limiting its utility. Defining MRSA positivity by PCR/ESI-TOF-MS is complicated by co-colonization of S. aureus with CoNS, which can also carry mecA.

Comparison of PCR/electron spray ionization-time-of-flight-mass spectrometry versus traditional clinical microbiology for active surveillance of organisms contaminating high-use surfaces in a burn intensive care unit, an orthopedic ward and healthcare workers.

BACKGROUND: Understanding nosocomial pathogen transmission is restricted by culture limitations. Novel platforms, such as PCR-based electron spray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS), may be useful as investigational tools. METHODS: Traditional clinical microbiology (TCM) and PCR/ESI-TOF-MS were used to recover and detect microorganisms from the hands and personal protective equipment of 10 burn intensive care unit (ICU) healthcare workers providing clinical care at a tertiary care military referral hospital. High-use environmental surfaces were assessed in 9 burn ICU and 10 orthopedic patient rooms. Clinical cultures during the study period were reviewed for pathogen comparison with investigational molecular diagnostic methods. RESULTS: From 158 samples, 142 organisms were identified by TCM and 718 by PCR/ESI-TOF-MS. The molecular diagnostic method detected more organisms (4.5 ± 2.1 vs. 0.9 ± 0.8, p < 0.01) from 99% vs. 67% of samples (p < 0.01). TCM detected S. aureus in 13 samples vs. 21 by PCR/ESI-TOF-MS. Gram-negative organisms were less commonly identified than gram-positive by both methods; especially by TCM. Among all detected bacterial species, similar percentages were typical nosocomial pathogens (18-19%) for TCM vs. PCR/ESI-TOF-MS. PCR/ESI-TOF-MS also detected mecA in 112 samples, vanA in 13, and KPC-3 in 2. MecA was associated (p < 0.01) with codetection of coagulase negative staphylococci but not S. aureus. No vanA was codetected with enterococci; one KPC-3 was detected without Klebsiella spp. CONCLUSIONS: In this pilot study, PCR/ESI-TOF-MS detected more organisms, especially gram-negatives, compared to TCM, but the current assay format is limited by the number of antibiotic resistance determinants it covers. Further large-scale assessments of PCR/ESI-TOF-MS for hospital surveillance are warranted.

Aminoglycoside resistance and susceptibility testing errors in Acinetobacter baumannii-calcoaceticus complex.

Antimicrobial resistance is depleting the pharmacopeia of agents clinically useful against Gram-negative bacilli. As the number of active agents diminishes, accurate susceptibility testing becomes critical. We studied the susceptibilities of 107 isolates of the Acinetobacter baumannii-calcoaceticus complex to amikacin, gentamicin, and tobramycin using disk diffusion, Etest, as well as the Phoenix, Vitek 2, and MicroScan automated systems, and compared the results to those obtained by broth microdilution. Genes encoding aminoglycoside-modifying enzymes (AMEs) were detected by multiplex PCR, and clonal relationships were determined by pulsed-field gel electrophoresis. Tobramycin was the most active aminoglycoside (27.1% of isolates were susceptible). Disk diffusion and Etest tended to be more accurate than the Vitek 2, Phoenix, and MicroScan automated systems; but errors were noted with all methods. The Vitek 2 instrument incorrectly reported that more than one-third of the isolates were susceptible to amikacin (a very major error). Isolates were polyclonal, with 26 distinct strains, and carried multiple AME genes unrelated to the strain type. The presence of the ant(2")-Ia gene was statistically associated with resistance to each aminoglycoside. The AME genotype accounted for the resistance profile observed in a minority of isolates, suggesting the involvement of multiple resistance mechanisms. Hospital pharmacy records indicated the preferential use of amikacin over other aminoglycosides in the burn intensive care unit, where aminoglycoside resistance is prevalent. The resistance in that unit did not correlate with a predominant strain, AME genotype, or total annual aminoglycoside consumption. Susceptibility to tobramycin increased, even though susceptible isolates carried AME genotypes predicting the inactivation of tobramycin. Determination of the relative contribution of multiple concurrent resistance mechanisms may improve our understanding of aminoglycoside resistance in the Acinetobacter baumannii-calcoaceticus complex.

Methicillin-resistant Staphylococcus aureus in wound cultures recovered from a combat support hospital in Iraq.

BACKGROUND: Staphylococcus aureus infections complicate care of combat-related injuries and can independently result in skin and soft-tissue infections during deployments or training. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains seem to produce severe disease but retain susceptibility to many oral antimicrobials. This study characterizes 84 MRSA isolates recovered from wound cultures at a combat support hospital in Iraq. METHODS: MRSA strains recovered from December 2007 through March 2009 were analyzed. Antimicrobial resistance testing was determined by broth microdilution and the BD Phoenix Automated Microbiology System. The genotypic pattern was analyzed by pulsed-field gel electrophoresis and polymerase chain reaction identification of resistance and virulence genes. RESULTS: No MRSA isolates from wound cultures were resistant to vancomycin. The most active oral antistaphylococcal agents were tetracycline (95% susceptibility), trimethoprim-sulfamethoxazole (94%), and clindamycin (94%). Of agents not typically recommended as monotherapy, 98% of isolates were susceptible to rifampin, 91% to moxifloxacin, and 60% to levofloxacin. The most common pulsed-field type (PFT) was USA300 (79%). The typical staphylococcal cassette chromosome mec IV elements carrying the CA-MRSA resistance genes were present in 88% of the isolates. Panton-Valentine leukocidin virulence genes were identified in 88% of isolates, including 100% of PFT USA300. The virulence gene associated with an arginine catabolic mobile element was present in 75% of isolates, including 94% of PFT USA300. CONCLUSION: This study is the first genotypic and phenotypic characterization of CA-MRSA recovered from wound cultures in a deployed combat hospital. The pattern noted was similar to that seen in soldiers stationed in the United States.

Thromboelastography as a better indicator of hypercoagulable state after injury than prothrombin time or activated partial thromboplastin time.

OBJECTIVES: To investigate the hemostatic status of critically ill, nonbleeding trauma patients. We hypothesized that a hypercoagulable state exists in patients early after severe injury and that the pattern of clotting and fibrinolysis are similar between burned and nonburn trauma patients. MATERIALS: Patients admitted to the surgical or burn intensive care unit within 24 hours after injury were enrolled. Blood samples were drawn on days 0 through 7. Laboratory tests included prothrombin time (PT), activated partial thromboplastin time (aPTT), levels of activated factor XI, D-dimer, protein C percent activity, antithrombin III percent activity, and thromboelastography (TEG). RESULTS: Study subjects were enrolled from April 1, 2004, to May 31, 2005, and included nonburn trauma patients (n = 33), burned patients (n = 25), and healthy (control) subjects (n = 20). Despite aggressive thromboprophylaxis, three subjects (2 burned and 1 nonburn trauma patients [6%]) had pulmonary embolism during hospitalization. Compared with controls, all patients had prolonged PT and aPTT (p < 0.05). The rate of clot formation (alpha angle) and maximal clot strength were higher for patients compared with those of controls (p < 0.05), indicating a hypercoagulable state. Injured patients also had lower protein C and antithrombin III percent activities and higher fibrinogen levels (p < 0.05 for all). Activated factor XI was elevated in 38% of patients (control subjects had undetectable levels). DISCUSSION: Thromboelastography analysis of whole blood showed that patients were in a hypercoagulable state; this was not detected by plasma PT or aPTT. The high incidence of pulmonary embolism indicated that our current prophylaxis regimen could be improved.

Acinetobacter skin colonization of US Army Soldiers.

OBJECTIVE: To evaluate whether skin colonization with Acinetobacter calcoaceticus-baumannii complex exists in a population of healthy, nondeployed US Army soldiers and, if present, how it might relate to the infections seen in current war casualties. DESIGN: We sampled various skin sites of soldiers to test for the presence of A. calcoaceticus-baumannii complex and to establish the prevalence of colonization. We then used ribotyping and antimicrobial susceptibility profiles to compare the isolates we recovered with A. calcoaceticus-baumannii complex isolates from injured soldiers. SETTING: Fort Sam Houston, Texas. PARTICIPANTS: A population of healthy, nondeployed US Army soldiers in training. RESULTS: A total of 17% of healthy soldiers were found to harbor A. calcoaceticus-baumannii complex. However, the strains differed from those recovered from injured soldiers. CONCLUSIONS: Skin carriage of A. calcoaceticus-baumannii complex exists among soldiers before deployment. However, the difference in the strains isolated from healthy soldiers, compared with the strains from injured soldiers, makes it difficult to identify skin colonization as the source of infection.

Bacterial Contamination of Burn Unit Employee Identity Cards.

The purpose of this study was to identify the presence or absence of pathogenic bacteria on burn intensive care unit employees' common access cards (CACs) and identity badges (IDs) and to identify possible variables that may increase risk for the presence of those bacteria. A prospective, cross-sectional study was conducted in our regional Burn Center in which bacterial swab specimens were collected from both the CAC and ID of 10 burn intensive care unit employees in each of five cohorts (nurses, respiratory therapists, physical therapists, physicians, and ancillary staff). Ten additional paired samples, collected from direct care staff in the outpatient burn clinic, served as control. Additional information described how the cards were worn and if/how they had been cleaned in the previous week. Fifty-eight CACs and 60 IDs were swabbed from participants. The overall contamination rate was 75%, with no trends identified based on how cards were worn. Bacteria were recovered from 86% (50/58) of CACs and 65% (39/60) of IDs, with CACs being significantly more contaminated overall than IDs (P < .01). In terms of potentially pathogenic bacteria, the overall rate was 3%, with 100% of those isolates coming from the outpatient clinic staff cohort (P < .001). When cleaned in the last week (n = 16), the contamination rate dropped to 50% overall (P = .003), indicating that even periodic cleaning appears to have a positive effect on bacterial contamination rates. The simple practice of routine identity card decontamination may reduce potential threats to patient safety as a result of nosocomial bacterial transmission.

[Release of DNA from Pseudomonas aeruginosa in vitro during spontaneous growth and treatment with ciprofloxacin].

OBJECTIVE: To observe the DNA release from Pseudomonas aeruginosa (P. aeruginosa) during spontaneous growth and exposure to different concentrations of ciprofloxacin (Cipro) in vitro. METHODS: The P. aeruginosa 1244 strain (ATCC 27317) was selected because it was sensitive to Cipro in vitro. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Cipro against this strain were determined, respectively. Different concentrations of Cipro were cultured with this strain at 37 degrees C for 4 h and 24 h. The samples of culture supernatant were filtered and electrophoresed in 1.8% agarose with SYBR Gold stain. Then the images of the gel sheets were photographed. RESULTS: The MIC and MBC of Cipro were 0.25 - 0.5 mg/L. The free bacterial DNA in 4 h culture samples with or without Cipro could not be detected by this method. The certain amount of free bacterial DNA molecules in 24 h culture samples without antibiotic appeared at the two zones whose molecular weights were more than 2000 bp and less than 100 bp. The large amount of free bacterial DNA molecules showed at three zones in 24 h culture samples with Cipro when its concentrations were much lower than its MIC. In terms of DNA molecular weight, the first two zones were above 2000 bp, and the third zone was below 100 bp. There was no detectable DNA release from bacteria in 24 h culture samples when Cipro was at or above its MIC. CONCLUSIONS: The certain amount of bacterial DNA were released from P. aeruginosa in the spontaneous growth. Different concentrations of Cipro had quite differential effects on the DNA release from P. aeruginosa in quantities and molecular weights in vitro.

Pathogens present in acute mangled extremities from Afghanistan and subsequent pathogen recovery.

Given the changing epidemiology of infecting pathogens in combat casualties, we evaluated bacteria and fungi in acute traumatic wounds from Afghanistan. From January 2013 to February 2014, 14 mangled lower extremities from 10 explosive-device injured casualties were swabbed for culture at Role 3 facilities. Bacteria were recovered from all patients on the date of injury. Pathogens recovered during routine patient care were recorded. The median injury severity score was 29, median initial Role 3/4 blood product support was 32 units, and median evacuation time was 42 minutes to first surgical care. Gram-positive bacteria were found in some wounds but not methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus. Most wounds were colonized with low-virulence, environmental gram-negative bacteria, and not recovered again during therapy, reflecting wound contamination. Only one wound had the same bacteria (E. cloacae) throughout care at the Role 3, 4, and 5 facilities. Three cultures from two patients had multidrug-resistant bacteria (E. cloacae, E. coli), all detected at Role 5 facilities. Molds were not detected at Role 3, whereas one patient had a mold at Role 4 and 5. Mangled lower extremity injuries have a high contamination rate with environmental organisms, which are not typically associated with infections during the course of the patient's care.

Comparison of Decontamination Methods for Human Skin Grafts.

Skin grafts intended for autologous transplant may be dropped on the operating room floor during handling. The authors examined optimal procedures for decontaminating tissue intended for burn surgery. Porcine skin (5 × 5 cm sections) harvested from expired animals using standard procedures was inoculated with either 10(6) CFU/ml Staphylococcus aureus or Klebsiella pneumoniae. Decontaminating strategies were compared: 10% povidone iodine, 0.04% chlorhexidine, or 50 U/ml bacitracin for injection, and mechanical agitation using normal saline or sterile water; each agent was applied for 60 seconds. Each skin section was blended and plated on agar for bacterial enumeration using the spread plate method. Tissue viability was evaluated in parallel using a cell viability reagent, along with a control (heat at 200 °C for 5 min). Bacterial counts were log transformed; one-way ANOVA with Tukey-Kramer HSD analysis were performed. Concentration of organisms <10(5) CFU/g was considered clinically insignificant colonization. Eight donors provided 21 S. aureus and six K. pneumoniae samples. After exposure, mean organism concentration (CFU/g) was <10(5) for povidone iodine (S. aureus 2.83 × 10(4); K. pneumoniae 1.85 × 10(4)), chlorhexidine (S. aureus 4.52 × 10(4); K. pneumoniae 1.77 × 10(4)), and normal saline (K. pneumoniae 8.76 × 10(4)) treated groups. After log transform, only povidone iodine and chlorhexidine were found to be different from control in both groups. Viability was decreased in the positive control group, but not in treatment groups. Agents routinely used for surgical skin prep (povidone iodine and chlorhexidine), reduced both Gram-positive and Gram-negative contamination in tissue intended for skin grafting procedures. Antiseptic treatments did not impair the cellular viability of porcine skin.

The bacterial contamination of surgical scrubs.

To our knowledge, no study has examined the bacterial profile of residents' scrubs. The goal of this investigation was to determine the bacterial profile of worn and unworn resident scrubs. Thirty pairs of scrubs were swabbed in 10 predetermined locations both prior to and after being worn continuously by the on-call resident. All swabs were screened for aerobic gram-positive and gram-negative bacteria. Bacteria underwent antimicrobial resistance testing and genetic relatedness by pulsed-field gel electrophoresis. Forty-one percent (123) of unworn scrub samples yielded bacteria, compared with 89% (268) of post-call scrub samples. On unworn scrubs, the most common organisms were coagulase-negative Staphylococcus (CNS; 94), gram positive rods (GPR; 34) and Streptococcus viridians (8). On post-call scrubs, the most common bacteria were CNS (271), micrococcus (51), Staphylococcus aureus (33), and GPR (28). All S. aureus were methicillin susceptible. There were different species, pulse-field types and antibiotic resistance profiles found amongst the CNS identified. No scrubs were found to harbor multidrug-resistant (MDR) organisms. This study found that unworn scrubs harbored normal skin flora and scrubs worn for at least 24 hours have a higher burden of bacteria than unworn scrubs but not an increased incidence of contamination with MDR organisms.

Evaluation of potential environmental contamination sources for the presence of multidrug-resistant bacteria linked to wound infections in combat casualties.

OBJECTIVE: To determine whether multidrug-resistant (MDR) gram-negative organisms are present in Afghanistan or Iraq soil samples, contaminate standard deployed hospital or modular operating rooms (ORs), or aerosolize during surgical procedures. DESIGN: Active surveillance. SETTING: US military hospitals in the United States, Afghanistan, and Iraq. METHODS: Soil samples were collected from sites throughout Afghanistan and Iraq and analyzed for presence of MDR bacteria. Environmental sampling of selected newly established modular and deployed OR high-touch surfaces and equipment was performed to determine the presence of bacterial contamination. Gram-negative bacteria aerosolization during OR surgical procedures was determined by microbiological analysis of settle plate growth. RESULTS: Subsurface soil sample isolates recovered in Afghanistan and Iraq included various pansusceptible members of Enterobacteriaceae, Vibrio species, Pseudomonas species, Acinetobacter lwoffii, and coagulase-negative Staphylococcus (CNS). OR contamination studies in Afghanistan revealed 1 surface with a Micrococcus luteus. Newly established US-based modular ORs and the colocated fixed-facility ORs revealed no gram-negative bacterial contamination prior to the opening of the modular OR and 5 weeks later. Bacterial aerosolization during surgery in a deployed fixed hospital revealed a mean gram-negative bacteria colony count of 12.8 colony-forming units (CFU)/dm(2)/h (standard deviation [SD], 17.0) during surgeries and 6.5 CFU/dm(2)/h (SD, 7.5; [Formula: see text]) when the OR was not in use. CONCLUSION: This study demonstrates no significant gram-negative bacilli colonization of modular and fixed-facility ORs or dirt and no significant aerosolization of these bacilli during surgical procedures. These results lend additional support to the role of nosocomial transmission of MDR pathogens or the colonization of the patient themselves prior to injury.

Activity of topical antimicrobial agents against multidrug-resistant bacteria recovered from burn patients.

BACKGROUND: Topical antimicrobials are employed for prophylaxis and treatment of burn wound infections despite no established susceptibility breakpoints, which are becoming vital in an era of multidrug-resistant (MDR) bacteria. We compared two methods of determining topical antimicrobial susceptibilities. METHODS: Isolates of Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae, and Acinetobacter baumanii-calcoaceticus (ABC) from burn patients were tested using broth microdilution and agar well diffusion to determine minimum inhibitory concentrations (MICs) and zones of inhibition (ZI). Isolates had systemic antibiotic resistance and clonality determined. MDR included resistance to antibiotics in three or more classes. RESULTS: We assessed 22 ESBL-producing K. pneumoniae, 20 ABC (75% MDR), 20 P. aeruginosa (45% MDR), and 20 MRSA isolates. The most active agents were mupirocin for MRSA and mafenide acetate for the gram-negatives with moderate MICs/ZI found with silver sulfadiazene, silver nitrate, and honey. MDR and non-MDR isolates had similar topical resistance. There was no clonality associated with resistance patterns. CONCLUSION: Despite several methods to test bacteria for topical susceptibility, no defined breakpoints exist and standards need to be established. We recommend continuing to use silver products for prophylaxis against gram-negatives and mafenide acetate for treatment, and mupirocin for MRSA.

Twenty-five year epidemiology of invasive methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered at a burn center.

Over the past two decades, an epidemiologic emergence of methicillin-resistant Staphylococcus aureus (MRSA) infections has occurred from that of primarily hospital-associated to community-associated. This emergence change has involved MRSA of different pulsed-field types (PFT), with different virulence genes and antimicrobial resistance patterns. In this study we, evaluate the changes in PFT and antimicrobial resistance epidemiology of invasive MRSA isolates over 25 years at a single burn unit. Isolates were tested by pulsed-field gel electrophoresis (PFGE), broth microdilution antimicrobial susceptibility testing, and PCR for the virulence factors Panton-Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME), and the resistance marker staphylococcal chromosomal cassette mec (SCCmec). Forty isolates were screened, revealing stable vancomycin susceptibility MIC without changes over time but decreasing susceptibility to clindamycin and ciprofloxacin. The majority of PFGE types were MRSA USA800 carrying the SCCmec I element and USA100 carrying the SCCmec II element. No strains typically associated with community-associated MRSA, USA300 or USA400, were found. USA800 isolates were predominately found in the 1980s, USA600 isolates were primarily found in the 1990s, and USA100 isolates were found in the 2000s. The PVL gene was present in only one isolate, the sole USA500 isolate, from 1987. The virulence marker ACME was not detected in any of the isolates. Overall, a transition was found in hospital-associated MRSA isolates over the 25 years, but no introduction of community-associated MRSA isolates into this burn unit. Continued active surveillance and aggressive infection control strategies are recommended to prevent the spread of community-acquired MRSA to this burn unit.

[Preliminary study on the release of DNA from Pseudomona aeruginosa induced by piperacillin/tazobactam in vitro].

OBJECTIVE: To observe the release of DNA from Pseudomonas aeruginosa (P. aeruginosa) induced by different concentrations of piperacillin/tazobactam (Piper) in vitro. METHODS: The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Piper against 1244 strain (ATCC 27317) of P. aeruginosa were determined, respectively. This strain of P. aeruginosa was separately cultured with Piper in different concentrations at 37 degrees C for 4 h and 24 h. The samples of cultural supernatant were filtered and electrophoresis was conducted in 1.8% agarose with SYBR Gold stain. Then the images of the gel sheets were photographed. RESULTS: This strain of P. aeruginosa was sensitive to Piper. The bacterial DNA was not detected in 4-h cultured P. aeruginosa either with or without Piper by this method. The bacterial DNA molecules could be detected in 24 h samples in cultures without Piper, and they were displayed in two zones of molecular weight over 2000 base pairs (bp) and lower than 100 bp. Similar results were observed when the MIC of piper (0.002, 0.004 g/L) were under the MIC measured at the 3rd time (0.008 g/L), but there was much more bacterial DNA with molecular weight lower than 100 bp. When Piper concentration was higher than its MIC, only smaller quantities of bacterial DNA in the area with molecular weight lower than 400 bp could be detected in 24-h culture samples. CONCLUSION: A certain amount of bacterial DNA was released from P. aeruginosa under its natural growth circumstance. Different concentrations of Piper showed different effects on DNA release, in regard to its quantity and molecular weight, from P. aeruginosa cultures.