Dynamics of colistin and tobramycin resistance among Enterobacter cloacae during prolonged use of selective decontamination of the digestive tract.

Background: A high prevalence of colistin resistance among E. cloacae isolates in two intensive care units (ICU) (of 16 and 6 beds) using selective digestive decontamination (SDD) since 1990 instigated a retrospective and prospective investigation to quantify the role of clonal transmission. SDD is topical application of colistin and tobramycin and systemic use of cefotaxime during the first days of ICU-admission. Methods: Multi-resistant E. cloacae (MREb) was defined as ESBL production and/or tobramycin non-susceptibility and/or colistin non-susceptibility. Incidence of acquisition and prevalence of carriage with MREb was determined from microbiological culture results. Results: Colistin-resistant E. cloacae was first detected in November 2009 and carriage was demonstrated in 141 patients until October 2014. Mean incidence of MREb acquisition was 4.61 and 1.86 per 1000 days at risk in ICUs 1 and 2, respectively, and the mean monthly prevalence of MREb in both ICUs was 7.0 and 3.1%, respectively, without a discernible trend in time. Conversion rates from carriage of colistin-susceptible to resistant E. cloacae were 0.20 and 0.13 per 1000 patient days, respectively. Whole genome sequencing of 149 isolates revealed eight clusters, with the number of SNPs of the largest two clusters ranging between 0 and 116 for cluster 1 (n = 49 isolates), and 0 and 27 for cluster 2 (n = 36 isolates), among isolates derived between 2009 and 2014. Conclusions: This study demonstrates a stable low-level endemicity of MREb in two Dutch ICUs with prolonged use of SDD, which was characterized by the persistent presence of two clusters, suggesting incidental clonal transmission.

Quantifying within-household transmission of extended-spectrum ß-lactamase-producing bacteria.

OBJECTIVES: Patients can acquire extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae during hospitalization, and colonized patients may transmit these bacteria after discharge, most likely to household contacts. In this study, ESBL transmission was quantified in households. METHODS: Faecal samples were longitudinally collected from hospitalized patients colonized with ESBL-producing bacteria and from their household members during hospitalization of the index patient and at 3, 6, 12 and 18 months. A mathematical household model was developed, which allowed for person-to-person transmission, acquisition from other sources (background transmission), and losing carriage. Next, a deterministic population model with a household structure was created, informed by parameter values found in the household model. RESULTS: In all, 74 index patients and 84 household members were included. In more than half of the household members ESBL-producing bacteria were demonstrated at some time during follow up. Person-to-person transmission occurred at a rate of 0.0053/colonized person/day (0.0025-0.011), background transmission at 0.00015/day (95% CI 0.00002-0.00039), and decolonization at 0.0026/day (0.0016-0.0040) for index patients and 0.0090/day (0.0046-0.018) for household members. The estimated probability of transmission from an index patient to a household contact was 67% and 37% vice versa. CONCLUSION: There is frequent transmission of ESBL-producing bacteria in households, which may contribute to the observed endemicity of ESBL carriage in the Netherlands. However, the population model suggests that there is not a single dominant acquisition route in the community.

Predicting carriage with extended-spectrum beta-lactamase-producing bacteria at hospital admission: a cross-sectional study.

The prevalence of patients colonized with extended-spectrum beta-lactamase (ESBL)-producing bacteria increases, especially in long-term-care facilities (LTCFs). Identification of ESBL carriers at hospital admission is relevant for infection control measures and antibiotic therapy for nosocomial infections. We aimed to develop a prediction rule for ESBL carriage at hospital admission for patients admitted from home and LTCFs, and to quantify incidences of nosocomial infections caused by ESBL-producing bacteria. The ESBL-carrier status was determined of patients admitted from LTCFs and from home settings in four hospitals in the Netherlands using perianal swabs obtained within 48 hours of admission. Risk factors for ESBL carriage were assessed. Infections caused by ESBL-producing bacteria were identified retrospectively. Among 1351 patients, 111 (8.2%) were ESBL carriers at admission: 50/579 (8.6%) admitted from LTCFs and 61/772 (7.9%) from home settings (p 0.63). Previous ESBL carriage and previous hospital admission were risk factors for ESBL carriage in multivariable analysis. The area under the curve of the receiver operating characteristic curve of the model was 0.64 (95% CI 0.58-0.71). Presence of ≥1 risk factor (n = 803; 59%) had sensitivity of 72%. Incidences of nosocomial infections caused by ESBL-producing bacteria were 45.5/10,000 and 2.1/10,000 admission days for ESBL carriers and non-carriers, respectively (p <0.05). In conclusion, prevalence of ESBL carriage at hospital admission was 8.2%, and was comparable among patients admitted from LTCF and home. A clinically useful prediction rule for ESBL carriage at admission could not be developed. The absolute incidence of nosocomial infections by ESBL-producing bacteria was low, but higher among patients carrying ESBL-producing bacteria at the time of hospital admission.

A disc diffusion assay for detection of class A, B and OXA-48 carbapenemases in Enterobacteriaceae using phenyl boronic acid, dipicolinic acid and temocillin.

Class A and B carbapenemases in Enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA, respectively. However, for OXA-48 (like) carbapenemases, no specific inhibitor is available. Because OXA-48 confers high-level temocillin resistance, a disc diffusion assay using temocillin as well as BA and DPA inhibition tests was evaluated for detection of class A, B and OXA-48 carbapenemases. The test collection included 128 well-characterized non-repeat Enterobacteriaceae isolates suspected of carbapenemase production; that is, with meropenem MICs ≥ 0.5 mg/L, including 99 carbapenemase producers (36 KPC, one GES, 31 MBL, four KPC plus VIM, 25 OXA-48, two OXA-162), and 29 ESBL and/or AmpC-producing isolates. PCR and sequencing of beta-lactamase genes was used as a reference test. Phenotypic carbapenemase detection was performed with discs (Rosco) containing meropenem (10 µg), temocillin (30 µg), meropenem + phenyl boronic acid (PBA), meropenem + DPA, meropenem + BA + DPA, and meropenem + cloxacillin (CL). Absence of synergy between meropenem and BA and/or DPA and a temocillin zone ≤10 mm was used to identify OXA-48. The sensitivity for identification of class A, B and OXA-48 carbapenemases was 95%, 90% and 100%, with 96-100% specificity. In non-Proteus species, the sensitivity for class B carbapenemase detection was 97%. All isolates without PBA or DPA synergy and a temocillin disc zone ≤10 mm were OXA-48 (like) positive. In conclusion, carbapenemase inhibition tests with PBA and DPA combined with a temocillin disc provide a reliable phenotypic confirmation method for class A, B and OXA-48 carbapenemases in Enterobacteriaceae.

Differences in the antibiotic susceptibility of human Escherichia coli with poultry-associated and non-poultry-associated extended-spectrum beta-lactamases.

The concurrent presence of bla CTX-M-1 and bla TEM-52 genes on similar plasmids of Escherichia coli isolated from poultry, chicken meat and humans supports the occurrence of food-borne transmission of extended-spectrum beta-lactamase (ESBL) genes. ESBL-producing E. coli (ESBL-E. coli) are most frequently detected in hospitalised patients and are known to spread in healthcare settings. We hypothesised that poultry-associated (PA) ESBL genes are predominant in the community, where acquisition is fuelled by food contamination, whereas non-PA ESBL genes are predominant in hospitals, with acquisition fuelled by cross-transmission. Then, differences in antimicrobial selective pressure in hospitals and poultry would create differences in co-resistance between PA and non-PA ESBL-E. coli. We, therefore, determined the prevalence and co-resistance of PA and non-PA ESBL-E. coli in community-acquired and nosocomial urinary tract infections in humans and bla CTX-M-1 and bla TEM-52 isolates from poultry. A total of 134 human ESBL-E. coli urine isolates were included in this study. Isolates containing bla CTX-M-1 or bla TEM-52 were considered to be PA, with the remainder being non-PA. Also, 72 poultry ESBL-E. coli were included. Minimum inhibitory concentration (MIC) values were determined by broth microdilution. The prevalence of PA ESBL genes in isolates obtained in general practice and hospitals was 28 % versus 30 % (n.s.). Human PA ESBL-E. coli were more frequently susceptible to ciprofloxacin (51 % vs. 25 %; p = 0.0056), gentamicin (86 % vs. 63 %; p = .0.0082), tobramycin (91 % vs. 34 %; p = 0.0001) and amikacin (98 % vs. 67 %; p = 0.0001) compared to human non-PA ESBL-E. coli. PA ESBL-E. coli are not more prevalent in community acquired than nosocomial urine samples, but are more often susceptible to ciprofloxacin and aminoglycosides than non-PA ESBL-E. coli. This does not support the existence of different reservoirs of ESBL genes.

Multi-centre evaluation of a phenotypic extended spectrum ß-lactamase detection guideline in the routine setting.

This study aimed to evaluate the routine setting performance of a guideline for phenotypic detection of extended spectrum ß-lactamases (ESBLs) in Enterobacteriaceae, recommending ESBL confirmation with Etest or combination disc for isolates with a positive ESBL screen test (i.e. cefotaxime and/or ceftazidime MIC >1 mg/L or an automated system ESBL warning). Twenty laboratories submitted 443 Enterobacteriaceae with a positive ESBL screen test and their confirmation test result (74%Escherichia coli, 12%Enterobacter cloacae, 8%Klebsiella pneumoniae, 3%Proteus mirabilis, 2%Klebsiella oxytoca). Presence of ESBL genes was used as reference test. Accuracy of local phenotypic ESBL detection was 88%. The positive predictive value (PPV) of local screen tests was 70%, and differed per method (Vitek-2: 69%, Phoenix: 68%, disc diffusion: 92%), and species (95%K. pneumoniae-27%K. oxytoca). A low PPV (3%) was observed for isolates with automated system alarm but third-generation cephalosporin MICs <2 mg/L. Local ESBL confirmation had a PPV and negative predictive value (NPV) of 93% and 90%, respectively. Compared with centrally performed confirmation tests, 7% of local tests were misinterpreted. Combination disc was more specific than Etest (91% versus 61%). Confirmation tests were not reliable for P. mirabilis and K. oxytoca (PPV 33% and 38%, respectively, although NPVs were 100%). In conclusion, performance of Etests could be enhanced by education of technicians to improve their interpretation, by genotypic ESBL confirmation of P. mirabilis and K. oxytoca isolates with positive phenotypic ESBL confirmation, and by interpreting isolates with a positive ESBL alarm but an MIC <2 mg/L for cefotaxime and ceftazidime as ESBL-negative.

Evaluation of a commercial microarray as a confirmation test for the presence of extended-spectrum ß-lactamases in isolates from the routine clinical setting.

Since the diagnostic characteristics of the Check-KPC ESBL microarray as a confirmation test on isolates obtained in a routine clinical setting have not been determined, we evaluated the microarray in a random selection of 346 clinical isolates with a positive ESBL screen test (MIC >1 mg/L for cefotaxime or ceftazidime or an ESBL alarm from the Phoenix or Vitek-2 expert system) collected from 31 clinical microbiology laboratories in the Netherlands in 2009. Using sequencing as the reference method the sensitivity of the microarray was 97% (237/245), the specificity 98% (97/99), the positive predictive value 99% (237/239) and the negative predictive value 92% (97/105).

Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains.

Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1) , bla(CTX-M-2) , bla(SHV-2) , bla(SHV-12) , bla(TEM-20) , bla(TEM-52) ): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.

Evaluation of the DiversiLab system for detection of hospital outbreaks of infections by different bacterial species.

Many bacterial typing methods are specific for one species only, time-consuming, or poorly reproducible. DiversiLab (DL; bioMérieux) potentially overcomes these limitations. In this study, we evaluated the DL system for the identification of hospital outbreaks of a number bacterial species. Appropriately typed clinical isolates were tested with DL. DL typing agreed with pulsed-field gel electrophoresis (PFGE) for Acinetobacter (n = 26) and Stenotrophomonas maltophilia (n = 13) isolates. With two exceptions, DL typing of Klebsiella isolates (n = 23) also correlated with PFGE, and in addition, PFGE-nontypeable (PFGE-NT) isolates could be typed. Enterobacter (n = 28) results also correlated with PFGE results; also, PFGE-NT isolates could be clustered. In a larger study (n = 270), a cluster of 30 isolates was observed that could be subdivided by PFGE. The results for Escherichia coli (n = 38) correlated less well with an experimental multilocus variable number of tandem repeats analysis (MLVA) scheme. Pseudomonas aeruginosa (n = 52) showed only a limited number of amplification products for most isolates. When multiple Pseudomonas isolates were assigned to a single type in DL, all except one showed multiple multilocus sequence types. Methicillin-resistant Staphylococcus aureus generally also showed a limited number of amplification products. Isolates that belonged to different outbreaks by other typing methods, including PFGE, spa typing, and MLVA, were grouped together in a number of cases. For Enterococcus faecium, the limited variability of the amplification products obtained made interpretation difficult and correlation with MLVA and esp gene typing was poor. All of the results are reflected in Simpson's index of diversity and adjusted Rand's and Wallace's coefficients. DL is a useful tool to help identify hospital outbreaks of Acinetobacter spp., S. maltophilia, the Enterobacter cloacae complex, Klebsiella spp., and, to a somewhat lesser extent, E. coli. In our study, DL was inadequate for P. aeruginosa, E. faecium, and MRSA. However, it should be noted that for the identification of outbreaks, epidemiological data should be combined with typing results.

Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis.

OBJECTIVES: Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. METHODS: Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). RESULTS: The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. CONCLUSIONS: This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

Decontamination of the digestive tract and oropharynx in ICU patients.

BACKGROUND: Selective digestive tract decontamination (SDD) and selective oropharyngeal decontamination (SOD) are infection-prevention measures used in the treatment of some patients in intensive care, but reported effects on patient outcome are conflicting. METHODS: We evaluated the effectiveness of SDD and SOD in a crossover study using cluster randomization in 13 intensive care units (ICUs), all in The Netherlands. Patients with an expected duration of intubation of more than 48 hours or an expected ICU stay of more than 72 hours were eligible. In each ICU, three regimens (SDD, SOD, and standard care) were applied in random order over the course of 6 months. Mortality at day 28 was the primary end point. SDD consisted of 4 days of intravenous cefotaxime and topical application of tobramycin, colistin, and amphotericin B in the oropharynx and stomach. SOD consisted of oropharyngeal application only of the same antibiotics. Monthly point-prevalence studies were performed to analyze antibiotic resistance. RESULTS: A total of 5939 patients were enrolled in the study, with 1990 assigned to standard care, 1904 to SOD, and 2045 to SDD; crude mortality in the groups at day 28 was 27.5%, 26.6%, and 26.9%, respectively. In a random-effects logistic-regression model with age, sex, Acute Physiology and Chronic Health Evaluation (APACHE II) score, intubation status, and medical specialty used as covariates, odds ratios for death at day 28 in the SOD and SDD groups, as compared with the standard-care group, were 0.86 (95% confidence interval [CI], 0.74 to 0.99) and 0.83 (95% CI, 0.72 to 0.97), respectively. CONCLUSIONS: In an ICU population in which the mortality rate associated with standard care was 27.5% at day 28, the rate was reduced by an estimated 3.5 percentage points with SDD and by 2.9 percentage points with SOD. (Controlled Clinical Trials number, ISRCTN35176830.)

[Optimalisation of the antibiotic policy in The Netherlands. XI. The national electronic antibiotic guide'SWAB-ID' for use in hospitals]. / Optimaliseren van het antibioticabeleid in Nederland. XI. Het nationale elektronische antibioticaboekje SWAB-ID voor ziekenhuizen.

The 'Stichting Werkgroep Antibioticabeleid' (Dutch Working Party on Antibiotic Policy) has developed an electronic national antibiotic guide for the antibiotic treatment and prophylaxis of common infectious diseases in hospitals. This guide also contains information on the most important characteristics of antimicrobial drugs. Advice on antibiotic treatment is based on existing national evidence-based guidelines, where available. Where no guideline is available, the advice is based on an inventory of the antibiotic policies of the 12 Dutch centres with an infectious disease or medical microbiology training programme. The national antibiotic guide can be accessed through the SWAB website (www.swab.nl) and can also be downloaded on PDA/PocketPC, free of charge. Every hospital antibiotic formulary committee in the Netherlands will be offered the opportunity to edit The national version for local use.

Presence of integron-associated resistance in the community is widespread and contributes to multidrug resistance in the hospital.

Integrons are strongly associated with the multidrug resistance seen in gram-negative bacilli in the hospital environment. No data, however, are available on their prevalence in the community. This study is the first to show that integrons are widespread in Enterobacteriaceae in the community and that integron-associated resistance genes in the community constitute a substantial reservoir for multidrug resistance in the hospital.

Novel gene cassettes and integrons.

An increase in multiresistant Enterobacteriaceae was observed at one of the departments of the University Medical Center Utrecht. Nine different integrons and 17 gene cassettes were found, including the new gene cassette aadA8. This cassette was highly related to aadA3 and aadA2. In addition, an unknown promoter sequence was found for two integrons.

Detection of Candida spp. in blood cultures using nucleic acid sequence-based amplification (NASBA).

Candida spp. are the main causes of fungal infections in immunocompromised patients. It is known, that the routinely used automated blood culture systems may fail to detect yeasts. We therefore investigated, whether Nucleic Acid Sequence-Based Amplification (NASBA) can be used to improve the detection rate of Candida spp. in blood cultures. Culture-positive as well as negative blood cultures from patients with a proven candidaemia were analyzed, and the results of BacT/Alert monitoring were compared with the results of NASBA-based detection of yeast RNA. With the NASBA-assay, the number of positive blood cultures increased from 21% to 34%. The NASBA-assay may confirm the diagnosis and demonstrate the need for prolonged treatment. In addition it may shorten the time to detection. In summary, using NASBA for the detection of yeast RNA in blood cultures, we have shown for the first time that it is possible to improve the detection rate of yeasts in blood cultures by using amplification technology.

Control of nosocomial multiresistant Enterobacteriaceae using a temporary restrictive antibiotic agent policy.

An observational study on the epidemiology of multiresistant Enterobacteriaceae was conducted in the neurology and neurosurgery wards of a university hospital to determine the impact of hospital hygiene measures and an additional temporary restrictive antibiotic agent policy on the sudden rise in incidence of these bacteria. The incidence and prevalence of patients with multiresistant Enterobacteriaceae were assessed, and patient isolates were typed phenotypically and by random amplified polymorphic DNA analysis. All hospital hygiene measures implemented were recorded, and the influence of the restrictive policy on antibiotic use was analyzed. This policy consisted of a prior authorization requirement and the withdrawal of all antibiotics with a possible selective pressure on multiresistant strains (gentamicin, tobramycin, quinolones, cotrimoxazole, broad-spectrum penicillins, and cephalosporins). This ban left only carbapenems and amikacin for treatment. Typing showed that 17 of the 61 (28%) patients involved were infected or colonized with a single multiresistant strain of Klebsiella oxytoca, for which an environmental source was identified. The isolates recovered from the other patients comprised eight different species, and subsequent genotyping yielded a great variety of strains. The increased incidence could not be controlled with hospital hygiene measures alone. Only after implementation of the restrictive antibiotic policy did the epidemic strain vanish and the endemic incidence of multiresistant Enterobacteriaceae decrease to <50% of the level before intervention. In the years since, the incidence has remained at this low level, and the antibiotic costs have decreased to a level lower than before intervention.

Severe neurological complications in association with Epstein-Barr virus infection.

Involvement of the nervous system in infectious mononucleosis is common. About 50% have headache on presentation. Neck stiffness without meningitis is a frequent finding. Severe neurological complications are rare though, occurring in fewer than 0.5%. We describe two patients with unusual and severe neurological complications in association with serological evidence of EBV-infection: a 32-year old female developed a bilateral optic neuritis combined with a transverse myelitis and a 72-year old man developed mononeuritis multiplex, autonomic neuropathy and a salt-wasting nephropathy.

Reinfection with Legionella pneumophila documented by pulsed-field gel electrophoresis.

A 50-year-old man with chronic lymphocytic leukemia experienced two episodes of community-acquired legionnaires' disease. For determining whether this was the result of relapse or reinfection, Legionella pneumophila strains, cultured from sputum specimens and his home water supply, were characterized by pulsed-field gel electrophoresis. Analysis of the restriction patterns and the clinical data indicated that the second disease period was most likely the result of reinfection. We conclude that after diagnosis of community-acquired legionnaires' disease in an immunocompromised patient, culturing of the domestic water supplies for L. pneumophila should be considered; if such cultures are positive, adequate measures to prevent reexposure have to be taken.