Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to β-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Cotransfer of antibiotic resistance genes and a hylEfm-containing virulence plasmid in Enterococcus faecium.

The hyl(Efm) gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hyl(Efm)-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hyl(Efm) gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hyl(Efm), whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hyl(Efm)-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hyl(Efm) plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

Emergence of methicillin-resistant Staphylococcus pseudintermedius in Switzerland: three cases of urinary tract infections in cats

Methicillin resistance has emerged in clinical isolates of Staphylococcus pseudintermedius from cats in Switzerland. Three cats suffering from urinary tract infections were infected with methicillin-resistant S. pseudintermedius (MRSP). Phenotypic and genotypic characterization of the resistance profile showed that the isolates displayed resistance to all beta-lactams and cephalosporins (blaZ, mecA), fluoroquinolones, tetracyclines [tet(K)], macrolides, lincosamides and streprogramins B [erm(B)], chloramphenicol (catpC221), trimethoprim [dfr(G)] and the aminoglycosides gentamicin [aac(6')-Ie-aph(2')-Ia], kanamycin and neomycin [aph(3')-III] and streptomycin [ant(6)-Ia]. They also harbor the leukocidin gene lukS-I. MRSP represents a new challenge for antibiotic therapy and this zoonotic bacteria may rapidly spread to animals and humans.

Antibiotic-induced expression of a cryptic cpb2 gene in equine beta2-toxigenic Clostridium perfringens

The cpb2 gene of beta2-toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non-porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non-porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full beta2-toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant beta2-toxin showed the absence of expression of the beta2-toxin in equine and the other non-porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the beta2-toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by beta2-toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic-induced ribosomal frameshifting.

Therapeutic drug monitoring of once daily aminoglycoside dosing: comparison of two methods and investigation of the optimal blood sampling strategy.

PURPOSE Therapeutic drug monitoring of patients receiving once daily aminoglycoside therapy can be performed using pharmacokinetic (PK) formulas or Bayesian calculations. While these methods produced comparable results, their performance has never been checked against full PK profiles. We performed a PK study in order to compare both methods and to determine the best time-points to estimate AUC0-24 and peak concentrations (C max). METHODS We obtained full PK profiles in 14 patients receiving a once daily aminoglycoside therapy. PK parameters were calculated with PKSolver using non-compartmental methods. The calculated PK parameters were then compared with parameters estimated using an algorithm based on two serum concentrations (two-point method) or the software TCIWorks (Bayesian method). RESULTS For tobramycin and gentamicin, AUC0-24 and C max could be reliably estimated using a first serum concentration obtained at 1 h and a second one between 8 and 10 h after start of the infusion. The two-point and the Bayesian method produced similar results. For amikacin, AUC0-24 could reliably be estimated by both methods. C max was underestimated by 10-20% by the two-point method and by up to 30% with a large variation by the Bayesian method. CONCLUSIONS The ideal time-points for therapeutic drug monitoring of once daily administered aminoglycosides are 1 h after start of a 30-min infusion for the first time-point and 8-10 h after start of the infusion for the second time-point. Duration of the infusion and accurate registration of the time-points of blood drawing are essential for obtaining precise predictions.

Nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) among Swiss veterinary health care providers: detection of livestock- and healthcare-associated clones.

We screened a total of 340 veterinarians (including general practitioners, small animal practitioners, large animal practitioners, veterinarians working in different veterinary services or industry), and 29 veterinary assistants for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) at the 2012 Swiss veterinary annual meeting. MRSA isolates (n = 14) were detected in 3.8 % (95 % CI 2.1 - 6.3 %) of the participants whereas MRSP was not detected. Large animal practitioners were carriers of livestock-associated MRSA (LA-MRSA) ST398-t011-V (n = 2), ST398-t011-IV (n = 4), and ST398-t034-V (n = 1). On the other hand, participants working with small animals harbored human healthcare-associated MRSA (HCA-MRSA) which belonged to epidemic lineages ST225-t003-II (n = 2), ST225-t014-II (n = 1), ST5-t002-II (n = 2), ST5-t283-IV (n = 1), and ST88-t186-IV (n = 1). HCA-MRSA harbored virulence factors such as enterotoxins, β-hemolysin converting phage and leukocidins. None of the MRSA isolates carried Panton-Valentine leukocidin (PVL). In addition to the methicillin resistance gene mecA, LA-MRSA ST398 isolates generally contained additional antibiotic resistance genes conferring resistance to tetracycline [tet(M) and tet(K)], trimethoprim [dfrK, dfrG], and the aminoglycosides gentamicin and kanamycin [aac(6')-Ie - aph(2')-Ia]. On the other hand, HCA-MRSA ST5 and ST225 mainly contained genes conferring resistance to the macrolide, lincosamide and streptogramin B antibiotics [erm(A)], to spectinomycin [ant(9)-Ia], amikacin and tobramycin [ant(4')-Ia], and to fluoroquinolones [amino acid substitutions in GrlA (S84L) and GyrA (S80F and S81P)]. MRSA carriage may represent an occupational risk and veterinarians should be aware of possible MRSA colonization and potential for developing infection or for transmitting these strains. Professional exposure to animals should be reported upon hospitalization and before medical intervention to allow for preventive measures. Infection prevention measures are also indicated in veterinary medicine to avoid MRSA transmission between humans and animals, and to limit the spread of MRSA both in the community, and to animal and human hospitals.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

Chromosomally and extrachromosomally mediated high-level gentamicin resistance in Streptococcus agalactiae

Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in non-pregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to loss of a synergistic effect. We therefore performed a multicentre study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centres in four countries, 1128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. Though, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon, designated Tn3706. In the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing a plasmid mediated HLGR in GBS. Thus, in our clinical GBS isolates HLGR is mediated both chromosomally and extrachromosomally.

Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection.

Aminoglycosides are commonly prescribed antibiotics with deleterious side effects to the inner ear. Due to their popular application as a result of their potent antimicrobial activities, many efforts have been undertaken to prevent aminoglycoside ototoxicity. Over the years, understanding of the antimicrobial as well as ototoxic mechanisms of aminoglycosides has increased. These mechanisms are reviewed in regard to established and potential future targets of hair cell protection.

Estado sanitário do jacaré-de-papo-amarelo (Caiman latirostris) em paisagens antropizadas no Estado de São Paulo

O jacaré-de-papo (Caiman latirostris) é considerado um crocodiliano de médio porte que apresenta uma ampla distribuição latitudinal na América do Sul. Possivelmente a espécie possui a situação mais complexa entre os crocodilianos brasileiros quanto ao aspecto da conservação, basicamente porque suas populações encontram-se fragmentadas em grande parte de sua área de distribuição original e por utilizarem áreas com fortes atividades antrópicas. Embora a espécie possua aparentemente um processo adaptativo frente a estas pressões, um aspecto fisiológico pode ainda sofrer rápida alteração quando submetido a elas: o estado sanitário. Desta maneira, este estudo objetivou determinar o estado sanitário do maior predador aquático, utilizando duas áreas distintas no Estado de São Paulo, através da determinação dos perfis hematológicos e bioquímicos do sangue, além da caracterização da microbiota oral, servindo de modelo à conservação da espécie em ambientes alterados. No primeiro capítulo foram determinados o perfil hematológico e bioquímico do sangue utilizando-se 29 indivíduos (19 machos e 10 fêmeas) capturados em Angatuba e 11 indivíduos (2 machos, 4 fêmeas e 5 filhotes) capturados em Cubatão. Diferenças estatísticas significativas foram encontradas nos valores de creatinina na comparação entre fêmeas de ambas as áreas de estudo (p = 0,033) e nos valores de alanina aminotransferase (p = 0,003), hemácias (p = 0,034), hemoglobina (p = 0,049) e volume corpuscular médio (p = 0,027) quando da comparação entre sexos. No segundo capítulo determinou-se a microbiota oral destes animais através do isolamento, identificação e caracterização bacteriana, em conjunto com o teste de perfil de susceptibilidade aos antimicrobianos. Foram determinados 14 diferentes tipos de bactérias na área de Angatuba, sendo uma classificada como baciliforme aeróbio Gram-positivo, uma como bacilo Gram-negativo e 12 classificadas como Enterobactérias Gram-negativas. Na área de Cubatão foram isolados cinco tipos de bactérias, sendo quatro classificadas como Enterobactérias Gram-negativas e uma como baciliforme aeróbio Gram-positivo. Considerando que o uso de antimicrobianos é um processo primário no tratamento de animais e pessoas, principalmente nos casos que envolvam infecções provocadas pela interação homem x animais, para ambas as áreas de estudo, as quinolonas Enrofloxacina e Norfloxacina, além do aminoglicosídeo Gentamicina, foram os antimicrobianos que apresentaram os menores índices de resistência frente aos isolados testados.

The use of a change in gentamicin clearance as an early predictor of gentamicin-induced nephrotoxicity

A retrospective review was undertaken in 744 patients who were dose-individualized with gentamicin once daily to evaluate a change in gentamicin clearance as a potential predictor of nephrotoxicity. The definition of nephrotoxicity was chosen to be a change in creatinine clearance greater than 20%. Similarly, a change in gentamicin clearance of greater than 20% was also considered a possible index of nephrotoxicity. Four criteria were developed to assess the usefulness of gentamicin clearance as a predictor of nephrotoxicity. Following the application of the inclusion/exclusion criteria, 132 patients were available for the analysis. The sensitivity, specificity, positive predictive value, and negative predictive value were assessed for each of the criteria. Receiver operating characteristic (ROC) curves were produced to determine if an optimum value in the change of gentamicin clearance could be found to maximize sensitivity and specificity. The overall incidence of nephrotoxicity based on a decrease in creatinine clearance by 20% or more was 3.8%. Women were overrepresented in the nephrotoxic group [71.4% versus 40.1% (P = 0.0025)]. Patients with nephrotoxicity had statistically longer treatment periods, increased cumulative dose, and more dosing predictions (P < 0.05 in each case). The sensitivity of the criteria ranged from 43 to 46%, and specificity ranged from 93 to 99%. The positive and negative predictive values ranged from 63 to 94% and 86 to 89%, respectively. In those patients in whom nephrotoxicity was predicted from a change in gentamicin clearance, this change occurred on average 3 days before the change in creatinine clearance (P < 0.05). A change in gentamicin clearance to predict nephrotoxicity may be a useful addition to current monitoring methods, although it is not the complete answer.

Antibacterial dosing in intensive care: Pharmacokinetics, degree of disease and pharmacodynamics of sepsis

Treatment of sepsis remains a significant challenge with persisting high mortality and morbidity. Early and appropriate antibacterial therapy remains an important intervention for such patients. To optimise antibacterial therapy, the clinician must possess knowledge of the pharmacokinetic and pharmacodynamic properties of commonly used antibacterials and how these parameters may be affected by the constellation of pathophysiological changes occurring during sepsis. Sepsis, and the treatment thereof, increases renal preload and, via capillary permeability, leads to 'third-spacing', both resulting in higher antibacterial clearances. Alternatively, sepsis can induce multiple organ dysfunction, including renal and/or hepatic dysfunction, causing a decrease in antibacterial clearance. Aminoglycosides are concentration-dependent antibacterials and they display an increased volume of distribution (V-d) in sepsis, resulting in decreased peak serum concentrations. Reduced clearance from renal dysfunction would increase the likelihood of toxicity. Individualised dosing using extended interval dosing, which maximises the peak serum drug concentration (C-max)/minimum inhibitory concentration ratio is recommended. beta-Lactams and carbapenems are time-dependent antibacterials. An increase in Vd and renal clearance will require increased dosing or administration by continuous infusion. If renal impairment occurs a corresponding dose reduction may be required. Vancomycin displays predominantly time-dependent pharmacodynamic properties and probably requires higher than conventionally recommended doses because of an increased V-d and clearance during sepsis without organ dysfunction. However, optimal dosing regimens remain unresolved. The poor penetration of vancomycin into solid organs may require alternative therapies when sepsis involves solid organs (e.g. lung). Ciprofloxacin displays largely concentration-dependent kill characteristics, but also exerts some time-dependent effects. The V-d of ciprofloxacin is not altered with fluid shifts or over time, and thus no alterations of standard doses are required unless renal dysfunction occurs. In order to optimise antibacterial regimens in patients with sepsis, the pathophysiological effects of systemic inflammatory response syndrome need consideration, in conjunction with knowledge of the different kill characteristics of the various antibacterial classes. In conclusion, certain antibacterials can have a very high V-d, therefore leading to a low C-max and if a high peak is needed, then this would lead to underdosing. The Vd of certain antibacterials, namely aminoglycosides and vancomycin, changes over time, which means dosing may need to be altered over time. Some patients with serum creatinine values within the normal range can have very high drug clearances, thereby producing low serum drug levels and again leading to underdosing. Copyright © 2010 Elsevier Inc. All rights reserved.

Renal Research

he author overviews two research projects in the School of Pharmacy at the University of Queensland. The first examines how GPs individualise drug doses with respect to renal function. The second looks at two different approaches to monitoring aminoglycoside antibiotics. (non-author abstract)

Dosing of gentamicin in patients with end-stage renal disease receiving hemodialysis

The aim of this study was to evaluate dosing schedules of gentamicin in patients with end-stage renal disease and receiving hemodialysis. Forty-six patients were recruited who received gentamicin while on hemodialysis. Each patient provided approximately 4 blood samples at various times before and after dialysis for analysis of plasma gentamicin concentrations. A population pharmacokinetic model was constructed using NONMEM (version 5). The clearance of gentamicin during dialysis was 4.69 L/h and between dialysis was 0.453 L/h. The clearance between dialysis was best described by residual creatinine clearance (as calculated using the Cockcroft and Gault equation), which probably reflects both lean mass and residual clearance mechanisms. Simulation from the final population model showed that predialysis dosing has a higher probability of achieving target maximum concentration (C-max) concentrations (> 8 mg/L) within acceptable exposure limits (area under the concentration-time curve [AUC] values > 70 and < 120 mg.h/L per 24 hours) than postdialysis dosing.