Katrin Mende

Biofilm formation by clinical isolates and the implications in chronic infections

BackgroundBiofilm formation is a major virulence factor contributing to the chronicity of infections. To date few studies have evaluated biofilm formation in infecting isolates of patients including both Gram-positive and Gram-negative multidrug-resistant (MDR) species in the context of numerous types of infectious syndromes. Herein, we investigated the biofilm forming capacity in a large collection of single patient infecting isolates and compared the relationship between biofilm formation to various strain characteristics.MethodsThe biofilm-forming capacity of 205 randomly sampled clinical isolates from patients, collected from various anatomical sites, admitted for treatment at Brooke Army Medical Center (BAMC) from 2004–2011, including methicillin-resistant/methicillin susceptible Staphylococcus aureus (MRSA/MSSA) (n=23), Acinetobacter baumannii (n=53), Pseudomonas aeruginosa (n=36), Klebsiella pneumoniae (n=54), and Escherichia coli (n=39), were evaluated for biofilm formation using the high-throughput microtiter plate assay and scanning electron microscopy (SEM). Relationships between biofilm formation to clonal type, site of isolate collection, and MDR phenotype were evaluated. Furthermore, in patients with relapsing infections, serial strains were assessed for their ability to form biofilms in vitro.ResultsOf the 205 clinical isolates tested, 126 strains (61.4%) were observed to form biofilms in vitro at levels greater than or equal to the Staphylococcus epidermidis, positive biofilm producing strain, with P. aeruginosa and S. aureus having the greatest number of biofilm producing strains. Biofilm formation was significantly associated with specific clonal types, the site of isolate collection, and strains positive for biofilm formation were more frequently observed to be MDR. In patients with relapsing infections, the majority of serial isolates recovered from these individuals were observed to be strong biofilm producers in vitro.ConclusionsThis study is the first to evaluate biofilm formation in a large collection of infecting clinical isolates representing diverse types of infections. Our results demonstrate: (1) biofilm formation is a heterogeneous property amongst clinical strains which is associated with certain clonal types, (2) biofilm forming strains are more frequently isolated from non-fluid tissues, in particular bone and soft tissues, (3) MDR pathogens are more often biofilm formers, and (4) strains from patients with persistent infections are positive for biofilm formation.

Infection-associated clinical outcomes in hospitalized medical evacuees after traumatic injury: trauma infectious disease outcome study.

Infections have long been known to complicate care in patients following traumatic injury frequently leading to excess morbidity and mortality.1, 2 In no setting is this more well-recognized than the challenging environment of combat casualty care. During the current military conflicts in Iraq and Afghanistan, Operations Iraqi and Enduring Freedom (OIF/OEF), major advances resulting in increased survival among wounded personnel have been observed. These include enhanced training of medics, forward deployment of surgical assets, rapid medical evacuation, and improvements in body armor.3–5 The significant advances leading to survival are coupled with major challenges in care due to the extensive nature of the injuries, profound bone and soft tissue disruption, and extensive wound contamination.6, 7 In addition, the rapid transit of these patients through multiple echelons of medical care places significant obstacles on infection control in an era of increasing risk due to hospital-associated multidrug resistant (MDRO) organisms.8, 9 The U.S. Department of Defense (DoD) has implemented a range of measures to improve combat casualty care and mitigate risk of infectious complications. A Joint Theater Trauma System and Joint Theater Trauma Registry (JTTR) have been developed to benchmark metrics and to provide a timely assessment of performance improvement interventions.5, 10, 11 Efforts to prevent infection include the development of guidelines for the prevention of infection related to combat injuries through comprehensive review of current evidence and consensus review by military and civilian experts in trauma, infectious disease, infection control, preventive medicine, and surgical specialties.12 In addition, standardized infection control measures across echelons of care accompanied by enhanced MDRO surveillance and serial evaluation have also been implemented.13, 14 Despite the growing literature describing infectious complications of combat-related trauma, there is still a lack of prospectively collected standardized infection data that includes specific therapy, microbiological findings and clinical outcomes across treatment facilities. This report describes the initial data and current status of an ongoing 5-year prospective observational cohort study of infectious complications associated with traumatic injury sustained during deployment, the DoD-Department of Veterans Affairs (VA) Trauma Infectious Disease Outcomes Study (TIDOS).

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.

Presence and Molecular Epidemiology of Virulence Factors in Methicillin-Resistant Staphylococcus aureus Strains Colonizing and Infecting Soldiers

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as an important cause of skin and soft-tissue infections (SSTI). The understanding of the molecular epidemiology and virulence of MRSA continues to expand. From January 2005 to December 2005, we screened soldiers for MRSA nasal colonization, administered a demographic questionnaire, and monitored them prospectively for SSTI. All MRSA isolates underwent molecular analysis, which included pulsed-filed gel electrophoresis (PFGE) and PCR for Panton-Valentine leukocidin (PVL), the arginine catabolic mobile element (ACME), and the staphylococcal cassette chromosome mec (SCCmec). Of the 3,447 soldiers screened, 134 (3.9%) had MRSA colonization. Of the 3,066 (89%) who completed the study, 39 developed culture-confirmed MRSA abscesses. Clone USA300 represented 53% of colonizing isolates but was responsible for 97% of the abscesses (P < 0.001). Unlike colonizing isolates, isolates positive for USA300, PVL, ACME, and type IV SCCmec were significantly associated with MRSA abscess isolates. As determined by multivariate analysis, risk factors for MRSA colonization were a history of SSTI and a history of hospitalization. Although various MRSA strains may colonize soldiers, USA300 is the most virulent when evaluated prospectively, and PVL, ACME, and type IV SCCmec are associated with these abscesses.

Recovery of Multidrug-Resistant Bacteria From Combat Personnel Evacuated From Iraq and Afghanistan at a Single Military Treatment Facility

U.S. combat casualties from Iraq and Afghanistan continue to develop infections with multidrug-resistant (MDR) bacteria. This study assesses the infection control database and clinical microbiology antibiograms at a single site from 2005 to 2007, a period when all Operation Iraqi Freedom (OIF)/Operation Enduring Freedom (OEF) casualties admitted to the facility underwent initial isolation and screening for MDR pathogens. During this 3-year period, there were 2,242 OIF/OEF admissions: 560 in 2005, 724 in 2006, and 958 in 2007. The most commonly recovered pathogens from OIF/OEF admission screening cultures were methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae and Acinetobacter. The yearly nosocomial infection rate of these three pathogens among OIF/OEF admissions ranged between 2 and 4%. There were remarkable changes in resistance profiles for Acinetobacter, K. pneumoniae, and S. aureus over time. Despite aggressive infection control procedures, there is continued nosocomial transmission within the facility and increasing antimicrobial resistance in some pathogens. Novel techniques are needed to control the impact of MDR bacteria in medical facilities.

Tetracycline Susceptibility Testing and Resistance Genes in Isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus Complex from a U.S. Military Hospital

ABSTRACT Infections with multidrug-resistant Acinetobacter baumannii-Acinetobacter calcoaceticus complex bacteria complicate the care of U.S. military personnel and civilians worldwide. One hundred thirty-three isolates from 89 patients at our facility during 2006 and 2007 were tested by disk diffusion, Etest, and broth microdilution for susceptibility to tetracycline, doxycycline, minocycline, and tigecycline. Minocycline was the most active in vitro, with 90% of the isolates tested susceptible. Susceptibilities varied significantly with the testing method. The acquired tetracycline resistance genes tetA, tetB, and tetA(39) were present in the isolates.

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

BackgroundThe 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.MethodsGNB 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.ResultsThe 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.ConclusionHealthy 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.

Antimicrobial Susceptibilities of Geographically Diverse Clinical Human Isolates of Leptospira

ABSTRACT Although antimicrobial therapy of leptospirosis has been studied in a few randomized controlled clinical studies, those studies were limited to specific regions of the world and few have characterized infecting strains. A broth microdilution technique for the assessment of antibiotic susceptibility has been developed at Brooke Army Medical Center. In the present study, we assessed the susceptibilities of 13 Leptospira isolates (including recent clinical isolates) from Egypt, Thailand, Nicaragua, and Hawaii to 13 antimicrobial agents. Ampicillin, cefepime, azithromycin, and clarithromycin were found to have MICs below the lower limit of detection (0.016 μg/ml). Cefotaxime, ceftriaxone, imipenem-cilastatin, penicillin G, moxifloxacin, ciprofloxacin, and levofloxacin had MIC90s between 0.030 and 0.125 μg/ml. Doxycycline and tetracycline had the highest MIC90s: 2 and 4 μg/ml, respectively. Doxycycline and tetracycline were noted to have slightly higher MICs against isolates from Egypt than against strains from Thailand or Hawaii; otherwise, the susceptibility patterns were similar. There appears to be possible variability in susceptibility to some antimicrobial agents among strains, suggesting that more extensive testing to look for geographic variability should be pursued.

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.

Carbapenem Susceptibility Testing Errors Using Three Automated Systems, Disk Diffusion, Etest, and Broth Microdilution and Carbapenem Resistance Genes in Isolates of Acinetobacter baumannii-calcoaceticus Complex

ABSTRACT The Acinetobacter baumannii-calcoaceticus complex (ABC) is associated with increasing carbapenem resistance, necessitating accurate resistance testing to maximize therapeutic options. We determined the accuracy of carbapenem antimicrobial susceptibility tests for ABC isolates and surveyed them for genetic determinants of carbapenem resistance. A total of 107 single-patient ABC isolates from blood and wound infections from 2006 to 2008 were evaluated. MICs of imipenem, meropenem, and doripenem determined by broth microdilution (BMD) were compared to results obtained by disk diffusion, Etest, and automated methods (the MicroScan, Phoenix, and Vitek 2 systems). Discordant results were categorized as very major errors (VME), major errors (ME), and minor errors (mE). DNA sequences encoding OXA beta-lactamase enzymes (blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like) and metallo-β-lactamases (MBLs) (IMP, VIM, and SIM1) were identified by PCR, as was the KPC2 carbapenemase gene. Imipenem was more active than meropenem and doripenem. The percentage of susceptibility was 37.4% for imipenem, 35.5% for meropenem, and 3.7% for doripenem. Manual methods were more accurate than automated methods. blaOXA-23-like and blaOXA-24-like were the primary resistance genes found. blaOXA-58-like, MBLs, and KPC2 were not present. Both automated testing and manual testing for susceptibility to doripenem were very inaccurate, with VME rates ranging between 2.8 and 30.8%. International variability in carbapenem breakpoints and the absence of CLSI breakpoints for doripenem present a challenge in susceptibility testing.