Tularemia in the Southeastern Swiss Alps at 1,700 m above sea level.

A 37-year-old man presented with a 4-day history of nonbloody diarrhea, fever, chills, productive cough, vomiting, and more recent sore throat. He worked for the municipality in a village in the Swiss Alps near St. Moritz. Examination showed fever (40 °C), hypotension, tachycardia, tachypnea, decreased oxygen saturation (90 % at room air), and bibasilar crackles and wheezing. Chest radiography and computed tomography scan showed an infiltrate in the left upper lung lobe. He responded to empiric therapy with imipenem for 5 days. After the imipenem was stopped, the bacteriology laboratory reported that 2/2 blood cultures showed growth of Francisella tularensis. He had recurrence of fever and diarrhea. He was treated with ciprofloxacin (500 mg twice daily, oral, for 14 days) and symptoms resolved. Further testing confirmed that the isolate was F. tularensis (subspecies holarctica) belonging to the subclade B.FTNF002-00 (Western European cluster). This case may alert physicians that tularemia may occur in high-altitude regions such as the Swiss Alps.

Antibiotic therapy for Klebsiella pneumoniae bacteremia: Implications of production of extended-spectrum beta-lactamases

The prevalence of extended-spectrum beta-lactamase (ESBL) production by Klebsiella pneumonia approaches 50% in some countries, with particularly high rates in eastern Europe and Latin America. No randomized trials have ever been performed on treatment of bacteremia due to ESBL-producing organisms; existing data comes only from retrospective, single-institution studies. In a prospective study of 455 consecutive episodes of Klebsiella pneumoniae bacteremia in 12 hospitals in 7 countries, 85 episodes were due to an ESBL-producing organism. Failure to use an antibiotic active against ESBL-producing K. pneumoniae was associated with extremely high mortality. Use of a carbapenem ( primarily imipenem) was associated with a significantly lower 14-day mortality than was use of other antibiotics active in vitro. Multivariate analysis including other predictors of mortality showed that use of a carbapenem during the 5-day period after onset of bacteremia due to an ESBL-producing organism was independently associated with lower mortality. Antibiotic choice is particularly important in seriously ill patients with infections due to ESBL-producing K. pneumoniae.

The postantibiotic effect of the carbapenem antibiotics on gram-negative bacteria

Postantibiotic effect (PAE) describes the suppression of microbial growth occurring after a short exposure to an antimicrobial agent. PAE appears to be a property of the majority of antimicrobial agents and is demonstrated by a wide variety of microorganisms. At present, carbapenems and penems are the only members of the -lactam group of antimicrobial agents that exhibit a significant PAE on Gram-negative bacilli. A standardised method was developed to evaluate the in vitro PAE of three carbapenems; imipenem, meropenem and biapenem on Gram-negative bacteria under reproducible laboratory conditions that partially mimicked those occurring in vivo. The effects on carbapenem PAE of the method of antimicrobial removal, concentration, exposure duration, inoculum size, inoculum growth phase, multiple exposures and pooled human serum were determined. Additionally, the reproducibility, susceptibility prior to and after PAE determination and inter-strain variation of carbapenem PAE were evaluated. The method developed determined PAE by utilising viable counts and demonstrated carbapenem PAE to be reproducible, constant over successive exposures, dependent on genera, concentration, duration of exposure, inoculum size and growth phase. In addition, carbapenem PAE was not significantly effected either by agitation, the antimicrobial removal method or the viable count diluent. At present, the mechanism underlying PAE is undetermined. It is thought to be due to either the prolonged persistence of the antimicrobial at the cellular site of action or the true recovery period from non-lethal damage. Increasing the L-lysine concentration and salinity at recovery decreased and increased the carbapenem and imipenem PAE of Pseudomonas aeruginosa, respectively. In addition, no apparent change was observed in the production of virulence factors by P.aeruginosa in PAE phase. However, alterations in cell morphology were observed throughout PAE phase, and the reappearance of normal cell morphology corresponded to the duration of PAE determined by viable count. Thus, the recovery of the penicillin binding protein target enzymes appears to be the mechanism behind carbapenem PAE in P. aeruginosa.

Antibiotic and biocide resistance in Salmonella enterica and Escherichia coli 0157

Bacterial resistance to antibiotics and biocides is a prevalent problem, which may be exacerbated by the commonplace and often unnecessary inclusion of biocides into domestic products. Addition of antimicrobials, to domestic disinfectants has raised concern about promoting microbial resistance and potential cross-resistance to therapeutic antibiotics. This study investigated the potential for resistance in Salmonella enterica serovars Enteritidis, Typhimurium, Virchow and Escherichia call 0157 to commonly used biocides, to identify mechanisms underlying resistance and whether these provided cross-resistance to antibiotics. Salmonella enterica and E. coli 0157 strains were serially exposed to sub-inhibitory. concentrations of erythromycin (ERY), benzalkonium chloride (BKC), chlorhexidine hydrochloride (CHX)and triclosan (TLN). Once resistance was achieved permeability changes in the outer membrane, including LPS, cell surface charge and hydrophobicityand the presence of,an active efflux were investigated as possible resistance candidates. Thin layer chromatography (TLC) and Gas chromatography (GC) were carried out to examine fatty acid and lipid changes in E. coli 0157 isolates with reduced susceptibility to TLN. Cross-resistance was studied by the Stoke's method and standard microdilution assays. Examination of the outer membrane proteins and LPS did not reveal any significant changes between parent and resistant strains. The hydrophobicity of the cells increased as the cells were passaged and became less. susceptible. An active efflux system was the most likely mechanism of resistance in all strains tested and a fab1 mutation was associated with E. coli 0157 resistant to TLN isolates. In all isolates investigated the resistance was stable for over 30 passages in biocide-free media. A high degree of cross-resistance was obtained in TLN-resjstant Escherichia coli 0157 strains, which repeatedly exerted decreased susceptibility to various antimicrobials, including chloramphenicol, erythromycin, imipenem, tetracycline and trimethoprirn:, as well as to various biocides. The results of this laboratory-based investigation suggest that it is possible for microorganisms to become resistant to biocides when repeatedly exposed to sublethal concentrations. This may be especially the case in the domestic environment where administration of biocides is poorly controlled. Eventually it could lead to the undesirable situation of resident strains becoming resistant to disinfection and cross resistant to other antimicrobials.

Studies of the nature of Burkholderia cepacia in cystic fibrosis

Burkholderia cepacia is an opportunistic pathogen that colonises of the lungs of cystic fibrosis (CF) patients, with a frequently fatal outcome. Antibiotic resistance is common and highly transmissible epidemic strains have been described in the UK. 37 B. cepacia isolates from clinical and botanical sources were characterised via metabolic capabilities, antibiotic sensitivity, fatty acid methyl ester (FAME) profiles restriction digest analysis of chromosomal DNA by pulsed-gel electrophoresis (PFGE) (with the use of two separate restriction enzymes) and outer membrane protein (OMP) profiles. This revealed isolates of the UK CF epidemic strain to form a distinct group with a specific OMP profile. Cluster analysis of PFGE and FAME profiles revealed the species Burkholderia gladioli and Burkholderia vietnamiensis to be more closely related to each other and to laboratory strains of B. cepacia than to the CF epidemic strain considered a member of the latter species. The epidemic strain of B. cepacia may therefore be worthy of species definition in its own right. All the strains studied showed a high level of resistance to antibiotics, including the carbapenems. Considering this, carbapenemase production by isolates of B. cepacia was investigated. A metallo-β-lactamase from a clinical strain of B. cepacia was isolated and partially purified of using Cibacron blue F3GA-coupled agarose. The resulting preparation showed a single band of β-lactamase activity (pI 8.45) after analytical isoelectric focusing. The enzyme was particularly effective in the hydrolysis of imipenem. Meropenem, biapenem, cephaloridine, ceftazidime, benzylpenicillin, ampicillin and carbenicillin were hydrolysed at a lower rate. An unusual inhibition profile was noted. Inhibition by the metal ion chelators ethylene diamine tetra acetic acid and o-phenanthroline was reversed by addition of zinc, indicating a metallo-enzyme, whilst >90% inhibition was attainable with 0.1mM concentrations of tazobactam and clavulanic acid. A study of 8 other clinical isolates showed an enzyme of pI 8.45 to be present and inducible by imipenem in each case. This enzyme was assigned PCM-I (Pseudomonas cepacia metalloenzyme I).

Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents

The mechanisms by which bacteria resist killing by antibiotics and biocides are still poorly defined, although repeated exposure to sublethal concentrations of antibacterial agents undoubtedly contributes to their development. This study aimed both to investigate the potential of Salmonella enterica and Escherichia coli O157 for adaptive resistance to commonly used biocides and to determine any cross-resistance to antibiotics. Strains were repeatedly passaged in media containing increasing concentrations of a biocide or antibiotic until adaptive resistance was obtained. A wide panel of antimicrobial agents was then screened by using the adapted strain to determine cross-resistance, if any. Adaptive resistance was readily achieved for both S. enterica and E. coli O157. Cross-resistance in adaptively resistant S. enterica varied with the serotype; Salmonella enterica serovar Enteritidis expressed cross-resistance to chloramphenicol, whereas Salmonella enterica serovar Typhimurium expressed cross-resistance to chlorhexidine. Benzalkonium chloride-resistant Salmonella enterica serovar Virchow showed elevated resistance to chlorhexidine; however, chlorhexidine-resistant Salmonella serovar Virchow did not demonstrate reciprocal cross-resistance to benzalkonium chloride, suggesting specific rather than generic resistance mechanisms. E. coli O157 strains acquired high levels of resistance to triclosan after only two sublethal exposures and, when adapted, repeatedly demonstrated decreased susceptibilities to various antimicrobial agents, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concern over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may contribute to the development of microbial resistance mechanisms.

Screening for AmpR-Specific Inhibitors to Combat P. aeruginosa Infections

Pseudomonas aeruginosa, a Gram-negative bacterium, an opportunistic pathogen that infects individuals suffering from reduced immunity or damaged tissue. The treatment of these infections has become a major problem due to its increasing antibiotic resistance. Many multi-drug resistant isolates of P. aeruginosa can thwart most antibiotic classes including ?- lactams, fluoroquinolones, and aminoglycosides. Its ability to combat ?-lactams is in part due to expression of AmpC, a major chromosomally encoded ?-lactamase. The expression of ampC is positively regulated by AmpR. Besides antibiotic resistance, AmpR is an important regulator of various factors that are required for establishing acute and chronic infections. Loss of ampR makes P. aeruginosa susceptible to ?-lactams and less virulent than the wild type. We hypothesize that AmpR is a potential therapeutic target. In the absence of new drugs in the pipeline, the aim of this study is to find an AmpR-specific inhibitor to assist and improve the use of currently available ?- lactam treatment. A small-molecule library from Torrey Pines Institute will be used in this study. Two reporter systems, lux and lacZ, fused to a PampC promotor will be used to assess AmpR activity. Positive hits will be those that inhibit 50% PampC activity in the presence of sub inhibitory concentration of imipenem, a ?- lactam. The top positive hits will be screened for their ability to cause human cell-cytotoxicity. The non-cytotoxic hits will be assessed for their ability to affect P. aeruginosa virulence and antibiotic resistance using various in vitro assays. Determination of potential AmpR inhibitors will prove to be useful in fighting off infections and may save countless patients suffering from these infections.

Perfis de resistência a agentes antimicrobianos de 5 estirpes do género Mycobacterium isoladas de ambiente Hospitalar

As infecções nosocomiais têm aumentado ao longo dos anos, resultando num aumento do tempo de permanência do doente no hospital, e permanecem como elevada causa de elevada morbilidade e mortalidade. As micobactérias são organismos que se encontram amplamente distribuídos no meio ambiente (M. mucogenicum, M. obuense e M. gordonae), incluindo, habitats marinhos (Mycobacterium marinum), sendo muitos deles patogénicos de mamíferos, e causadores de diferentes patologias, como a Lepra e a Tuberculose. M. marinum causa uma doença sistémica tal como tuberculose em peixes e pode causar infecções da pele em seres humanos (Granuloma de Aquário) que se podem propagar para estruturas mais profundas como ossos (osteomielite). Enquanto que M. obuense é causador de infecções do tracto respiratório, M. mucogenicum e M. gordonae promovem bacteremias. Este estudo teve como principal objectivo a identificação das populações bacterianas e o seu isolamento, em particular micobactérias ambientais em dois hospitais, que sabe serem responsáveis, cada vez mais por infecções atípicas como bacteremias (M. mucogenicum e M. gordonae), infecções pulmonares (M. obuense) e infecções cutâneas (M. marinum). Pretendeu-se também avaliar a resistência aos antibióticos e desinfectantes comummente utilizados no tratamento de infecções causadas por micobactérias não tuberculosas (MNT) através do cálculo da Concentração Mínima Inibitória (CMI) para aferir os perfis de resistência. Os resultados deste estudo demonstram a identificação de 186 espécies de bactérias em dois hospitais amostrados das quais se identificaram 5 estirpes de micobactérias – “M. gardonae” (10AIII, 29AIII e 35AIII), “M. obuense” (22DIII) e “M. mucogenicum” (24AIII). Das 5 estirpes de micobactérias identificadas “M. gardonae” 10AIII apresenta perfil de resistência ao imipenemo (CMI = 16 mg/L); “M. gardonae” 29AIII apresenta perfil de resistência à claritromicina (CMI = 8 mg/L) e “M. gardonae” 35AIII apresenta, por sua vez, apenas perfil de susceptibilidade intermédia ao imipenem (CMI = 8 mg/L). M. obuense 22DIII apresenta perfil de resistência ao imipenem (CMI = 32 mg/L), à tobramicina (CMI=32 mg/L) e à ciprofloxacina (CMI = 8 mg/L). “M. mucogenicum” apresenta perfil de resistência ao sulfametoxazol (CMI > 128 mg/L), à doxiciclina (CMI>64 mg/L), à tobramicina (CMI=16 mg/L) e à ciprofloxacina (CMI=4 mg/L).Em conclusão pôde-se verificar que além da presença de um grande leque de bactérias capazes de causar infecções nosocomiais nos hospitais, MNT também existem na forma multirresistente, o que revela uma problemática a ter em atenção. Esta requer mais estudo dos mecanismos de resistência e da sua disseminação, e obtenção de novos medicamentos com novos alvos, mais eficazes para combater as estirpes multirresistentes que ao longo dos anos tem aumentado.

Multi-drug resistant Staphylococcus aureus isolated from emergency ward of an Iranian hospital

Purpose: To study the prevalence of resistant strains of Staphylococcus aureus isolated from surfaces, beds and various equipment of an Iranian hospital emergency ward. Methods: Two hundred swab samples were collected from the surfaces, beds, trolleys, surgical equipment and diagnostic medical devices in emergency ward. Samples were cultured and those that were S. aureus-positive were confirmed using polymerase chain reaction (PCR). Antimicrobial resistance pattern was analyzed using disk diffusion method. Results: Nine of 200 samples (4.5 %) collected were positive for S. aureus. Surfaces (8.8 %), beds (5 %) and trolleys (5 %) were the most commonly contaminated. S. aureus isolates exhibited varying levels of resistance against antibiotics with the following being the highest: tetracycline (88.8 %), penicillin (88.8 %) and ampicillin (77.7 %). The prevalence of resistance against methicillin, oxacillin and azithromycin were 44.4, 33.3 and 33.3 %, respectively. There was no pattern of resistance against imipenem. Conclusion: Efficient disinfection of surfaces, beds, trolleys and surgical instruments should be performed periodically to reduce colonization of resistant strains of S. aureus in various areas of emergency health care centers.