Characterization of Neisseria gonorrhoeae isolates detected in Switzerland (1998–2012): emergence of multidrug-resistant clones less susceptible to cephalosporins

Background: The spread of Neisseria gonorrhoeae (Ng) isolates resistant to the clinically implemented antibiotics is challenging the efficacy of treatments. Unfortunately, phenotypic and molecular data regarding Ng detected in Switzerland are scarce. Methods: We compared the characteristics of Ng detected during 1998–2001 (n = 26) to those detected during 2009–2012 (n = 34). MICs were obtained with the Etest and interpreted as non-susceptible (non-S) according to EUCAST criteria. Sequence type (ST) was achieved implementing the NG-MAST. BlaTEM, ponA, penA, mtrR, penB, tet (M), gyrA, parC, mefA, ermA/B/C/F, rplD, rplV, and 23S rRNA genes were analyzed. Results: The following susceptibility results were obtained (period: % of non-S, MIC90 in mg/L): penicillin (1998–2001: 42.3%, 3; 2009–2012: 85.3%, 16), cefixime (1998–2001: 0%, ≤0.016; 2009–2012: 8.8%, 0.125), ceftriaxone (1998–2001: 0%, 0.004; 2009–2012: 0%, 0.047), ciprofloxacin (1998–2001: 7.7%, 0.006; 2009–2012: 73.5%, ≥32), azithromycin (1998–2001: 11.5%, 0.25; 2009–2012: 23.6%, 0.38), tetracycline (1998–2001: 65.4%, 12; 2009–2012: 88.2%, 24), spectinomycin (1998–2001: 0%, 12; 2009–2012: 0%, 8). The prevalence of multidrug-resistant (MDR) isolates increased from 7.7% in 1998–2001 to 70.6% in 2009–2012. International STs and genogroups (G) emerged during 2009–2012 (G1407, 29.4%; G2992, 11.7%; G225, 8.8%). These isolates possessed distinctive mechanisms of resistance (e.g., G1407: PBP1 with L421, PBP2 pattern XXXIV, GyrA with S91F and D95G, ParC with S87R, PorB with G120K and A121N, mtrR promoter with A deletion). Conclusions: The prevalence of penicillin- ciprofloxacin- and tetracycline-resistant Ng has reached dramatic levels, whereas cefixime and ceftriaxone show MICs that tend to increase during time. International MDR clones less susceptible to cephalosporins are rapidly emerging indicating that the era of untreatable gonococcal infections is close.

Determination of an inoculum effect with various cephalosporins among clinical isolates of methicillin-susceptible Staphylococcus aureus.

Using 98 clinical methicillin-susceptible Staphylococcus aureus isolates of known beta-lactamase (Bla) type, we found a pronounced inoculum effect for cephalexin (mostly Bla type A and C strains), a mild inoculum effect for cephalothin (especially types B and C), and no inoculum effects for ceftriaxone and cefuroxime. Ceftobiprole showed the lowest MICs at a high inoculum but with a slight increase for Bla-positive versus Bla-negative strains. Since a potential therapeutic effect associated with a cephalosporin inoculum effect has been described, further studies are warranted.

Impact of the use of β-lactam antimicrobials on the emergence of Escherichia coli isolates resistant to cephalosporins under standard pig-rearing conditions

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained blaCTX-M-1, 10 contained blaCTX-M-14, 4 contained blaCTX-M-9, 2 contained blaCTX-M-15, and 5 contained blaSHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals.

Rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae by use of the new chromogenic βLacta test

The chromogenic βLacta test developed for the rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae revealed good performance with extended-spectrum β-lactamase (ESBL) producers (97.5% true-positive results). However, false-negative results occurred with chromosomal AmpC hyperproducers and plasmid AmpC producers, whereas uninterpretable results were mostly due to VIM-1 carbapenemase producers and possibly low levels of expressed ESBLs.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

First detection of extended-spectrum cephalosporin- and fluoroquinolone-resistant Escherichia coli in Australian food-producing animals

This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n = 324) from Australian food-producing animals [cattle (n = 169), porcine (n = 114), poultry (n = 32) and sheep (n = 9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0–88.6%), ampicillin (9.4–71.1%), trimethoprim/sulfamethoxazole (11.1–67.5%) and streptomycin (21.9–69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of blaCMY-2. This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages.

Expressão de fatores de virulência, mecanismos de resistência aos agentes antimicrobianos e análise molecular da resistência aos beta-lactâmicos de enterobactérias isoladas de bolsas periodontais

Em estudo anterior, as espécies de enterobactérias apresentando perfis variados de resistência aos antimicrobianos foram detectadas em 20% dos sítios com lesões periodontais de pacientes sadios do ponto de vista sistêmico. Tais cepas microbianas foram submetidas a investigações com o intuito de determinar à expressão de enzimas hidrolíticas para substratos diversos, a multirresistência aos agentes antimicrobianos e os mecanismos de resistência aos antimicrobianos da classe dos β lactâmicos. A maioria das amostras expressou atividade de gelatinase (65%), caseinase (30%) e elastase (10%). Lipase, lecitinase e DNase foram observadas apenas para Serratia marcescens. A multirresistência (considerado como a resistência a pelo menos dois agentes antimicrobianos de famílias diferentes) foi observada em 56% das amostras isoladas. A maioria das cepas foi resistentes à ampicilina (93,75%) e amoxicilina/ácido clavulânico (81,25%). Investigações sobre a resistência aos antibióticos β-lactâmicos mostraram que três amostras resistentes à cefalosporinas de 2 geração, apresentaram perfis plasmidiais de diferentes pesos moleculares. A expressão fenotípica de β-lacatamases, foi detectada nas cepas de Enterobacter cloacae (PcOM46 e PcOM5) e S. marcescens (PcOM63). No entanto, na análise molecular, não foi possível confirmar a expressão fenotípica de diferentes β-lactamases, com exceção do E. cloacae PcOM46, que apresentou amplificação para AmpC e blaTEM. Embora sensível à maioria dos antibióticos β-lactâmicos (exceção feita à ampicilina e amoxicilina / ácido clavulânico), amostra de S. marcescens PcOM68 apresentou amplificação para o gene blaSHV. Os experimentos de conjugação não detectaram a transferência de plasmídios para uma cepa de Escherichia coli K12 sensívei aos β-lactâmicos, o mesmo ocorreu nos procedimentos de transformação por eletroporação e por CaCl2, sugerindo uma resistência dependente de genes cromossomiais. A expressão de diferentes atividades enzimáticas, juntamente com a resistência aos antimicrobianos, aponta estes grupos de bactérias como agentes patogênicos potenciais capazes de contribuir para a patogênese e resposta à quimioterapia antimicrobiana nas doenças periodontais, além da disseminação sistêmica para outros locais do corpo, especialmente em indivíduos imunocomprometidos. A colonização prévia de lesões periodontais por espécies resistentes aos β-lactâmicos, pode contribuir para a disseminação destes genes relacionados à resistência aos antimicrobianos em ambientes hospitalares.

Análise da prevalência de resistência aos antimicrobianos em pacientes com infecção urinária comunitária de um laboratório privado na cidade do Rio de Janeiro

Descrever a prevalência das espécies bacterianas isoladas nas infecções urinárias comunitárias. Descrever os perfis de susceptibilidade aos antibióticos de uso oral utilizado frente às bactérias isoladas nas infecções urinárias comunitárias. Avaliar a prevalência de fenótipos de resistência bacterianos através dos resultados dos testes de susceptibilidade e dos rastreamentos específicos utilizados. Amostras colhidas exclusivamente no atendimento ambulatorial com contagens de unidades formadoras de colônias entre 100.000 a ≥1.000.000 por mililitro (UCF/ml) Com ou sem piúria no exame de elementos anormais na urina e sedimentoscopia (EAS). Foram analisados retrospectivamente os resultados de urinoculturas e dos testes de susceptibilidade a antimicrobianos, realizados em um Laboratório da rede privada na cidade do Rio de janeiro, de pacientes atendidos em ambulatórios e com quadros de ITU. As amostras de urina coletadas englobavam basicamente os seguintes bairros: Botafogo, Barra da Tijuca, Ipanema, Copacabana, Tijuca e Centro. Foram analisados um total de 8.475 culturas de urina divididas em 7.286 urinas de pacientes femininos e 1.189 de pacientes masculinos entre Janeiro de 2006 a Dezembro de 2012. As amostras foram todas coletadas na Cidade do Rio de Janeiro e englobavam basicamente os seguintes bairros: Botafogo, Barra da Tijuca, Ipanema, Copacabana, Tijuca e Centro. Encontramos um percentual de resistência de 27% para ciprofloxacina frente à Escherichia coli que com 68.23% é a principal etiologia encontrada na ITU na comunidade os resultados das três fluoroquinolonas avaliadas no estudo, ciprofloxacina (2 geração), levofloxacina (3 geração) e norfloxacina (2 geração), acharemos respectivamente 27%, 25% e 20% de resistência em Escherichia coli. O uso de fluoroquinolonas em infecções urinárias comunitárias e consequentemente os achados de padrões de resistência neste estudo, reforçam o que já foi descrito em outros trabalhos. A cefalosporina de 2 geração (cefuroxima), demonstrou percentuais de resistência bastante satisfatórios frente as principais etiologias. Em Escherichia coli o percentual foi de 2%, em Klebsiella pneumoniae 3% e em Proteus mirabilis não houve nenhum achado de resistência. Uma das vantagens da cefuroxima é ser ativa quanto à produção de beta lactamase, conferindo um espectro maior frente a possíveis produtoras desta enzima. Seu esquema posológico é de 250mg duas vezes ao dia por 7 dias para infecções urinárias não complicadas. O meio mais eficaz de melhorar a administração antimicrobiana provavelmente envolverá um programa abrangente que incorpora múltiplas estratégias e colaboração entre as diversas especialidades dentro de uma determinada instituição de saúde. Neste contexto, a observação periódica da incidência bacteriana com seus respectivos índices de resistência aos antimicrobianos por sitio de infecção e correlação com os antibióticos mais comumente utilizados, é mandatória para o sucesso terapêutico.

Evolução da resistência aos antibióticos β-lactâmicos em Staphylococcus aureus

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Integrated analysis of CANVAS 1 and 2: phase 3, multicenter, randomized, double-blind studies to evaluate the safety and efficacy of ceftaroline versus vancomycin plus aztreonam in complicated skin and skin-structure infection.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of complicated skin and skin-structure infection (cSSSI). Increasing antimicrobial resistance in cSSSI has led to a need for new safe and effective therapies. Ceftaroline was evaluated as treatment for cSSSI in 2 identical phase 3 clinical trials, the pooled analysis of which is presented here. The primary objective of each trial was to determine the noninferiority of the clinical cure rate achieved with ceftaroline monotherapy, compared with that achieved with vancomycin plus aztreonam combination therapy, in the clinically evaluable (CE) and modified intent-to-treat (MITT) patient populations. METHODS: Adult patients with cSSSI requiring intravenous therapy received ceftaroline (600 mg every 12 h) or vancomycin plus aztreonam (1 g each every 12 h) for 5-14 days. RESULTS: Of 1378 patients enrolled in both trials, 693 received ceftaroline and 685 received vancomycin plus aztreonam. Baseline characteristics of the treatment groups were comparable. Clinical cure rates were similar for ceftaroline and vancomycin plus aztreonam in the CE (91.6% vs 92.7%) and MITT (85.9% vs 85.5%) populations, respectively, as well as in patients infected with MRSA (93.4% vs 94.3%). The rates of adverse events, discontinuations because of an adverse event, serious adverse events, and death also were similar between treatment groups. CONCLUSIONS: Ceftaroline achieved high clinical cure rates, was efficacious against cSSSI caused by MRSA and other common cSSSI pathogens, and was well tolerated, with a safety profile consistent with the cephalosporin class. Ceftaroline has the potential to provide a monotherapy alternative for the treatment of cSSSI. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT00424190 for CANVAS 1 and NCT00423657 for CANVAS 2.

Eradication of endotracheal tube biofilm by nebulised gentamicin.

Objective: To compare the efficacy of gentamicin, nebulised via the endotracheal tube (ET), with that of parenteral cefotaxime or parenteral cefuroxime in preventing the formation of ET biofilm.

Setting: General intensive care units in two university teaching hospitals.

Design: The microbiology of ET biofilm from 36 ICU patients eligible to receive antibiotic prophylaxis was examined. Peak and trough tracheal concentrations of gentamicin, cefotaxime or cefuroxime were measured in each patient group, on the 2nd day of intubation.

Patients: Twelve patients received gentamicin (80 mg) nebulised in 4 ml normal saline every 8 h, 12 cefotaxime (1 g, 12 hourly) and 12 cefuroxime (750 mg, 8 hourly). Prophylaxis was continued for the duration of intubation.

Measurements and results: Samples of tracheal secretions were taken on the 2nd day of ventilation for determination of antibiotic concentrations. Following extubation, ETs were examined for the presence of biofilm. Pathogens considered to be common aetiological agents for VAP included Staphylococcus aureus, enterococci, Enterobacteriaceae and pseudomonads. While microbial biofilm was found on all ETs from the cephalosporin group, microbial biofilm of these micro-organisms was found on 7 of the 12 ET tubes from patients receiving cefotaxime [S. aureus (4), pseudomonads (1), Enterobacteriaceae (1), enterococcus (1)] and 8 of the 12 ET tubes from patients receiving cefuroxime [Enterobacteriaceae (6), P. aeruginosa (1) and enterococcus (1)]. While microbial biofilm was observed on five ETs from patients receiving nebulised gentamicin, none of these were from pathogens for ventilator-associated pneumonia (VAP). Tracheal concentrations of both cephalosporins were lower than those needed to inhibit the growth of pathogens recovered from ET tube biofilm. The median (and range) concentrations for cefotaxime were 0.90 (<0.23–1.31) mg/l and 0.28 (<0.23–0.58) mg/l for 2 h post-dose and trough samples, respectively. Two hours post-dose concentrations of cefuroxime (median and range) were 0.40 (0.34–0.83) mg/l, with trough concentrations of 0.35 (<0.22–0.47) mg/l. Tracheal concentrations (median and range) of gentamicin measured 1 h post-nebulisation were 790 (352–>1250) mg/l and then, before the next dose, were 436 (250–1000) mg/l.

Conclusion: Nebulised gentamicin attained high concentrations in the ET lumen and was more effective in preventing the formation of biofilm than either parenterally administered cephalosporin and therefore may be effective in preventing this complication of mechanical ventilation.

Modelling the impact of antibiotic use and infection control practices on the incidence of hospital-acquired methicillin-resistant Staphylococcus aureus: a time-series analysis

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide. A wide range of factors have been suggested to influence the spread of MRSA. The objective of this study was to evaluate the effect of antimicrobial drug use and infection control practices on nosocomial MRSA incidence in a 426-bed general teaching hospital in Northern Ireland.

Methods: The present research involved the retrospective collection of monthly data on the usage of antibiotics and on infection control practices within the hospital over a 5 year period (January 2000–December 2004). A multivariate ARIMA (time-series analysis) model was built to relate MRSA incidence with antibiotic use and infection control practices.

Results: Analysis of the 5 year data set showed that temporal variations in MRSA incidence followed temporal variations in the use of fluoroquinolones, third-generation cephalosporins, macrolides and amoxicillin/clavulanic acid (coefficients = 0.005, 0.03, 0.002 and 0.003, respectively, with various time lags). Temporal relationships were also observed between MRSA incidence and infection control practices, i.e. the number of patients actively screened for MRSA (coefficient = -0.007), the use of alcohol-impregnated wipes (coefficient = -0.0003) and the bulk orders of alcohol-based handrub (coefficients = -0.04 and -0.08), with increased infection control activity being associated with decreased MRSA incidence, and between MRSA incidence and the number of new patients admitted with MRSA (coefficient = 0.22). The model explained 78.4% of the variance in the monthly incidence of MRSA.

Conclusions: The results of this study confirm the value of infection control policies as well as suggest the usefulness of restricting the use of certain antimicrobial classes to control MRSA.

Role of AcrR and ramA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore

The MICs of ciprofloxacin for 33 clinical isolates of K. pneumoniae resistant to extended-spectrum cephalosporins from three hospitals in Singapore ranged from 0.25 to >128 microg/ml. Nineteen of the isolates were fluoroquinolone resistant according to the NCCLS guidelines. Strains for which the ciprofloxacin MIC was >or=0.5 microg/ml harbored a mutation in DNA gyrase A (Ser83-->Tyr, Leu, or IIe), and some had a secondary Asp87-->Asn mutation. Isolates for which the MIC was 16 microg/ml possessed an additional alteration in ParC (Ser80-->IIe, Trp, or Arg). Tolerance of the organic solvent cyclohexane was observed in 10 of the 19 fluoroquinolone-resistant strains; 3 of these were also pentane tolerant. Five of the 10 organic solvent-tolerant isolates overexpressed AcrA and also showed deletions within the acrR gene. Complementation of the mutated acrR gene with the wild-type gene decreased AcrA levels and produced a two- to fourfold reduction in the fluoroquinolone MICs. None of the organic solvent-tolerant clinical isolates overexpressed another efflux-related gene, acrE. While marA and soxS were not overexpressed, another marA homologue, ramA, was overexpressed in 3 of 10 organic solvent-tolerant isolates. These findings indicate that multiple target and nontarget gene changes contribute to fluoroquinolone resistance in K. pneumoniae. Besides AcrR mutations, ramA overexpression (but not marA or soxS overexpression) was related to increased AcrAB efflux pump expression in this collection of isolates.

ß-Lactams: chemical structure, mode of action and mechanisms of resistance

This synopsis summarizes the key chemical and bacteriological characteristics of β-lactams, penicillins, cephalosporins, carbanpenems, monobactams and others. Particular notice is given to first-generation to fifth-generation cephalosporins. This review also summarizes the main resistance mechanism to antibiotics, focusing particular attention to those conferring resistance to broad-spectrum cephalosporins by means of production of emerging cephalosporinases (extended-spectrum β-lactamases and AmpC β-lactamases), target alteration (penicillin-binding proteins from methicillin-resistant Staphylococcus aureus) and membrane transporters that pump β-lactams out of the bacterial cell.