In Vitro activity of Manuka Honey and polyhexamethylene biguanide on filamentous fungi and toxicity to human cell lines.

Soft-tissue invasive fungal infections are increasingly recognized as significant entities directly contributing to morbidity and mortality. They complicate clinical care, requiring aggressive surgical debridement and systemic antifungal therapy. To evaluate new topical approaches to therapy, we examined the antifungal activity and cytotoxicity of Manuka Honey (MH) and polyhexamethylene biguanide (PHMB). The activities of multiple concentrations of MH (40%, 60%, 80%) and PHMB (0.01%, 0.04%, 0.1%) against 13 clinical mould isolates were evaluated using a time-kill assay between 5 min and 24 h. Concentrations were selected to represent current clinical use. Cell viability was examined in parallel for human epidermal keratinocytes, dermal fibroblasts and osteoblasts, allowing determination of the 50% viability (LD50) concentration. Antifungal activity of both agents correlated more closely with exposure time than concentration. Exophiala and Fusarium growth was completely suppressed at 5 min for all PHMB concentrations, and at 12 and 6 h, respectively, for all MH concentrations. Only Lichtheimia had persistent growth to both agents at 24 h. Viability assays displayed concentration-and time-dependent toxicity for PHMB. For MH, exposure time predicted cytotoxicity only when all cell types were analyzed in aggregate. This study demonstrates that MH and PHMB possess primarily time-dependent antifungal activity, but also exert in vitro toxicity on human cells which may limit clinical use. Further research is needed to determine ideal treatment strategies to optimize antifungal activity against moulds while limiting cytotoxicity against host tissues in vivo.

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.

In vitro activity of Melaleuca alternifolia (tea tree) oil on filamentous fungi and toxicity to human cells.

Invasive fungal wound infections (IFIs) are increasingly reported in trauma patients and cause considerable morbidity and mortality despite standard of care treatment in trauma centers by experienced medical personnel. Topical agents such as oil of melaleuca, also known as tea tree oil (TTO), have been proposed for adjunctive treatment of IFIs. We evaluated the activity of TTO against filamentous fungi associated with IFIs by testing 13 clinical isolates representing nine species via time-kill assay with seven concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, and 1%). To ascertain the safety of topical application to wounds, cell viability assays were performed in vitro using human fibroblasts, keratinocytes, osteoblasts, and umbilical vein endothelial cells with 10 concentrations of TTO (75%, 50%, 25%, 10%, 5%, and 10-fold serial dilutions from 1 to 0.0001%) at five time points (5, 15, 30, 60, and 180 min). Compatibility of TTO with explanted porcine tissues was also assessed with eight concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0.1%) at the time points used for cellular assays and at 24 h. The time-kill studies showed that fungicidal activity was variable between isolates. The effect of TTO on cell viability was primarily concentration dependent with significant cytotoxicity at concentrations of ≥ 10% and ≥ 50% for cells lines and whole tissue, respectively. Our findings demonstrate that TTO possesses antifungal activity against filamentous fungi associated with IFIs; furthermore that negligible effects on whole tissues, in contrast to individual cells, were observed following exposure to TTO. Collectively, these findings indicate a potential use of TTO as topical treatment of IFIs.

Clinical infectious outcomes associated with biofilm-related bacterial infections: a retrospective chart review.

BACKGROUND: Biofilms are associated with persistent infection. Reports characterizing clinical infectious outcomes and patient risk factors for colonization or infection with biofilm forming isolates are scarce. Our institution recently published a study examining the biofilm forming ability of 205 randomly selected clinical isolates. This present study aims to identify potential risk factors associated with these isolates and assess clinical infectious outcomes. METHODS: 221 clinical isolates collected from 2005 to 2012 and previously characterized for biofilm formation were studied. Clinical information from the associated patients, including demographics, comorbidities, antibiotic usage, laboratory values, and clinical infectious outcomes, was determined retrospectively through chart review. Duplicate isolates and non-clinical isolates were excluded from analysis. Associations with biofilm forming isolates were determined by univariate analysis and multivariate analysis. RESULTS: 187 isolates in 144 patients were identified for analysis; 113 were biofilm producers and 74 were not biofilm producers. Patients were primarily male (78 %) military members (61 %) with combat trauma (52 %). On multivariate analysis, the presence of methicillin-resistant Staphylococcus aureus (p < 0.01, OR 5.09, 95 % CI 1.12, 23.1) and Pseudomonas aeruginosa (p = 0.02, OR 3.73, 95 % CI 1.46, 9.53) were the only characteristics more likely to be present in the biofilm producing isolate group. Infectious outcomes of patients with non-biofilm forming isolates, including cure, relapse/reinfection, and chronic infection, were similar to infectious outcomes of patients with biofilm-forming isolates. Mortality with initial infection was higher in the biofilm producing isolate group (16 % vs 5 %, p = 0.01) but attributable mortality was low (1 of 14). No characteristics examined in this study were found to be associated with relapse/reinfection or chronic infection on multivariate analysis. CONCLUSIONS: Bacteria species, but not clinical characteristics, were associated with biofilm formation on multivariate analysis. Biofilm forming isolates and non-biofilm forming isolates had similar infectious outcomes in this study.

Phenotypic and genotypic changes over time and across facilities of serial colonizing and infecting Escherichia coli isolates recovered from injured service members.

Escherichia coli is the most common colonizing and infecting organism isolated from U.S. service members injured during deployment. Our objective was to evaluate the phenotypic and genotypic changes of infecting and colonizing E. coli organisms over time and across facilities to better understand their transmission patterns. E. coli isolates were collected via surveillance cultures and infection workups from U.S. military personnel injured during deployment (June 2009 to May 2011). The isolates underwent antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and multiplex PCR for phylotyping to determine their resistance profiles and clonality. A total of 343 colonizing and 136 infecting E. coli isolates were analyzed, of which 197 (57%) and 109 (80%) isolates, respectively, produced extended-spectrum ß-lactamases (ESBL). Phylogroup A was predominant among both colonizing (38%) and infecting isolates (43%). Although 188 unique pulsed-field types (PFTs) were identified from the colonizing isolates, and 54 PFTs were identified from the infecting isolates, there was a lack of PFT overlap between study years, combat zones, and military treatment facilities. On a per-subject basis, 26% and 32% of the patients with serial colonizing isolates and 10% and 21% with serial infecting isolates acquired changes in their phylogroup and PFT profiles, respectively, over time. The production of ESBL remained high over time and across facilities, with no substantial changes in antimicrobial susceptibilities. Overall, our results demonstrated an array of genotypic and phenotypic differences for the isolates without large clonal clusters; however, the same PFTs were occasionally observed in the colonizing and infecting isolates, suggesting that the source of infections may be endogenous host organisms.

Biofilms and persistent wound infections in United States military trauma patients: a case-control analysis.

BACKGROUND: Complex traumatic injuries sustained by military personnel, particularly when involving extremities, often result in infectious complications and substantial morbidity. One factor that may further impair patient recovery is the persistence of infections. Surface-attached microbial communities, known as biofilms, may play a role in hindering the management of infections; however, clinical data associating biofilm formation with persistent or chronic infections are lacking. Therefore, we evaluated the production of bacterial biofilms as a potential risk factor for persistent infections among wounded military personnel. METHODS: Bacterial isolates and clinical data from military personnel with deployment-related injuries were collected through the Trauma Infectious Disease Outcomes Study. The study population consisted of patients with diagnosed skin and soft-tissue infections. Cases (wounds with bacterial isolates of the same organism collected 14 days apart) were compared to controls (wounds with non-recurrent bacterial isolates), which were matched by organism and infectious disease syndrome. Potential risk factors for persistent infections, including biofilm formation, were examined in a univariate analysis. Data are expressed as odds ratios (OR; 95% confidence interval [CI]). RESULTS: On a per infected wound basis, 35 cases (representing 25 patients) and 69 controls (representing 60 patients) were identified. Eight patients with multiple wounds were utilized as both cases and controls. Overall, 235 bacterial isolates were tested for biofilm formation in the case-control analysis. Biofilm formation was significantly associated with infection persistence (OR: 29.49; CI: 6.24-infinity) in a univariate analysis. Multidrug resistance (OR: 5.62; CI: 1.02-56.92), packed red blood cell transfusion requirements within the first 24 hours (OR: 1.02; CI: 1.01-1.04), operating room visits prior to and on the date of infection diagnosis (OR: 2.05; CI: 1.09-4.28), anatomical location of infected wound (OR: 5.47; CI: 1.65-23.39), and occurrence of polymicrobial infections (OR: 69.71; CI: 15.39-infinity) were also significant risk factors for persistent infections. CONCLUSIONS: We found that biofilm production by clinical strains is significantly associated with the persistence of wound infections. However, the statistical power of the analysis was limited due to the small sample size, precluding a multivariate analysis. Further data are needed to confirm biofilm formation as a risk factor for persistent wound infections.

Biofilm formation by clinical isolates and the implications in chronic infections.

BACKGROUND: Biofilm 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. METHODS: The 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. RESULTS: Of 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. CONCLUSIONS: This 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.

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.

Variations of CHROMagar Acinetobacter to detect imipenem-resistant Acinetobacter baumannii-calcoaceticus complex.

BACKGROUND: Acinetobacter baumannii-calcoaceticus complex (ABC) isolates are often multidrug-resistant, including to carbapenems. Chromogenic media can facilitate the rapid detection of Gram-negative bacteria, often with the addition of supplements to a base chromogenic medium to detect resistance. We examined various combinations of available media to detect imipenem resistance among 107 ABC clinical isolates. METHODS: CHROMagar Orientation, CHROMagar KPC, and CHROMagar Acinetobacter, by itself, with Acinetobacter supplement, with KPC supplement, or CHROMagar Acinetobacter with increasing concentrations (1, 2.5, and 5 ml/l) of a new CR102 supplement, were examined. RESULTS: Sensitivity for the detection of isolates was high (> 98%) for all formulations. Specificity was high for CHROMagar Acinetobacter with 2.5 ml/l and 5 ml/l of the CR102 supplement, at 95.3% and 97.7%, respectively, with positive predictive values of 97% and 98.5%. Negative predictive values of these 2 formulations were 100%. CONCLUSIONS: CHROMagar Acinetobacter with the addition of the CR102 supplement at 2.5 ml/l and 5ml/l is highly sensitive and specific for the detection of imipenem-resistant ABC, and may be useful for the rapid detection of imipenem-resistant ABC in clinical samples.

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.

Effect of HMG-CoA reductase inhibitors on antimicrobial susceptibilities for gram-negative rods.

Epidemiologic evidence suggests a beneficial effect of HMG-CoA reductase inhibitors (statins) in sepsis, and in-vitro data exist for antimicrobial activity of statins against some bacteria and fungi. We examined whether statin exposure at physiologic concentrations enhances activity of selected antimicrobials against Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Broth microdilution was performed with and without dose-ranging concentrations of lovastatin, fluvastatin, atorvastatin, pravastatin and simvastatin. No effects on antimicrobial activity were demonstrated.

Clinical Characteristics and Resistance Patterns of Pseudomonas aeruginosa Isolated From Combat Casualties.

INTRODUCTION: Multidrug-resistant (MDR) Gram-negative infections complicate care of combat casualties. We describe the clinical characteristics, resistance patterns, and outcomes of Pseudomonas aeruginosa infections in combat casualties. METHODS: Combat casualties included in the Trauma Infectious Disease Outcomes Study with infections with and without P. aeruginosa isolation during initial hospitalization were compared. Pseudomonas aeruginosa from initial wound, blood, and serial isolates (≥7 days from previous isolate) collected from June 2009 through February 2014 was subjected to antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and whole genome sequencing for assessing clonality. Multidrug resistance was determined using the CDC National Healthcare Safety Network definition. RESULTS: Of 829 combat casualties with infections diagnosed during initial hospitalization, 143 (17%) had P. aeruginosa isolated. Those with P. aeruginosa were more severely injured (median Injury Severity Score 33 [interquartile range (IQR) 27-45] vs 30 [IQR 18.5-42]; P < .001), had longer hospitalizations (median 58.5 [IQR 43-95] vs 38 [IQR 26-56] days; P < .001), and higher mortality (6.9% vs 1.5%; P < .001) than those with other organisms. Thirty-nine patients had serial P. aeruginosa isolation (median 2 subsequent isolates; IQR: 1-5), with decreasing antimicrobial susceptibility. Ten percent of P. aeruginosa isolates were MDR, associated with prior exposure to antipseudomonal antibiotics (P = .002), with amikacin and colistin remaining the most effective antimicrobials. Novel antimicrobials targeting MDR Gram-negative organisms were also examined, and 100% of the MDR P. aeruginosa isolates were resistant to imipenem/relabactam, while ceftazidime/avibactam and ceftolozane/tazobactam were active against 35% and 56% of the isolates, respectively. We identified two previously unrecognized P. aeruginosa outbreaks involving 13 patients. CONCLUSIONS: Pseudomonas aeruginosa continues to be a major cause of morbidity, affecting severely injured combat casualties, with emergent antimicrobial resistance upon serial isolation. Among MDR P. aeruginosa, active antimicrobials remain the oldest and most toxic. Despite ongoing efforts, outbreaks are still noted, reinforcing the crucial role of antimicrobial stewardship and infection control.

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.

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 (bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), and bla(OXA-51-like)) and metallo-ß-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. bla(OXA-23-like) and bla(OXA-24-like) were the primary resistance genes found. bla(OXA-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.

Oral antibiotics for infections due to multidrug-resistant Gram-negative organisms.

We determined minimum inhibitory concentrations of rifampicin, nitrofurantoin, amoxicillin-clavulanic acid, and cefdinir, plus a combination of amoxicillin-clavulanic acid and cefdinir by broth microdilution for mainly wound isolates of Escherichia coli and Klebsiella pneumoniae. E. coli and K. pneumoniae susceptibilities increased by combining amoxicillin-clavulanic acid and cefdinir.

Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive Klebsiella variicola in trauma patients.

Recent reclassification of the Klebsiella genus to include Klebsiella variicola, and its association with bacteremia and mortality, has raised concerns. We examined Klebsiella spp. infections among battlefield trauma patients, including occurrence of invasive K. variicola disease. Klebsiella isolates collected from 51 wounded military personnel (2009-2014) through the Trauma Infectious Disease Outcomes Study were examined using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis. K. variicola isolates were evaluated for hypermucoviscosity phenotype by the string test. Patients were severely injured, largely from blast injuries, and all received antibiotics prior to Klebsiella isolation. Multidrug-resistant Klebsiella isolates were identified in 23 (45%) patients; however, there were no significant differences when patients with and without multidrug-resistant Klebsiella were compared. A total of 237 isolates initially identified as K. pneumoniae were analyzed, with 141 clinical isolates associated with infections (remaining were colonizing isolates collected through surveillance groin swabs). Using PCR sequencing, 221 (93%) isolates were confirmed as K. pneumoniae, 10 (4%) were K. variicola, and 6 (3%) were K. quasipneumoniae. Five K. variicola isolates were associated with infections. Compared to K. pneumoniae, infecting K. variicola isolates were more likely to be from blood (4/5 versus 24/134, p = 0.04), and less likely to be multidrug-resistant (0/5 versus 99/134, p<0.01). No K. variicola isolates demonstrated the hypermucoviscosity phenotype. Although K. variicola isolates were frequently isolated from bloodstream infections, they were less likely to be multidrug-resistant. Further work is needed to facilitate diagnosis of K. variicola and clarify its clinical significance in larger prospective studies.

Tetracycline susceptibility testing and resistance genes in isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex from a U.S. military hospital.

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.

Antimicrobial susceptibilities of geographically diverse clinical human isolates of Leptospira.

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 microg/ml). Cefotaxime, ceftriaxone, imipenem-cilastatin, penicillin G, moxifloxacin, ciprofloxacin, and levofloxacin had MIC(90)s between 0.030 and 0.125 microg/ml. Doxycycline and tetracycline had the highest MIC(90)s: 2 and 4 microg/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.

Low Prevalence of carbapenem-resistant Enterobacteriaceae among wounded military personnel.

Multidrug-resistant organisms (MDROs) are a global health problem that affect both civilian and military populations. Among wounded warriors, MDROs further complicate the care of trauma-related infections, resulting in extended duration of hospitalization, as well as increased morbidity and mortality. During the wars in Iraq and Afghanistan, extended spectrum ß-lactamase-producing Enterobacteriaceae were frequently isolated from wounded warriors. The potential emergence of difficult-to-treat carbapenem-resistant Enterobacteriaceae represented a serious challenge for clinicians. We examined carbapenem-resistant Enterobacteriaceae prevalence among wounded military personnel over a 6-year period (2009-2015). Among 4090 Enterobacteriaceae isolates collected, 16 (0.4%) were carbapenem-resistant, of which the majority was Enterobacter aerogenes (44%) followed by Klebsiella pneumoniae (37%), and Escherichia coli (19%). Five isolates (31%) collected from 2 patients were carbapenemase-producers with one associated with an infection. All 5 carbapenemase-producing isolates were resistant to all tested carbapenems and each carried one carbapenemase gene (4 with blaKPC-3 and 1 with blaNDM-1). Overall, although a large number of Enterobacteriaceae isolates were collected, only a small proportion was carbapenem-resistant and data indicate a lack of a cluster. Due to these limited numbers, it is difficult to make any conclusions regarding the association between carbapenem resistance, antibiotic exposure, and clinical outcomes.