C. Velasco

Plasmid-mediated quinolone resistance: an update

In 1998, the first plasmid-mediated gene involved in quinolone resistance (currently named qnrA1) was reported. Extra qnr-like plasmid-mediated genes (qnrB, qnrS, qnrC, qnrD) and their chromosomal homologues have also been characterized. These genes code for a pentapeptide repeat protein that protects type II topoisomerases from quinolones. Since then, there have been reports of two other plasmid-mediated resistance mechanisms: the modification of quinolones with a piperazinyl substituent by the acetyltransferase, Aac(6′)-Ib-cr; and active efflux by QepA and OqxAB, pumps related to major facilitator superfamily (MFS) transporters. These genes have a wide geographic distribution (mainly in Enterobacteriaceae). Because of the difficulties of phenotypic detection of this type of resistance, its real prevalence is only partially known. One important point is that although these mechanisms cause only low-level resistance, they favor and complement the selection of other resistance mechanisms.

Impact of an Evidence-Based Bundle Intervention in the Quality-of-Care Management and Outcome of Staphylococcus aureus Bacteremia

BACKGROUND Staphylococcus aureus bacteremia (SAB) is associated with significant morbidity and mortality. Several aspects of clinical management have been shown to have significant impact on prognosis. The objective of the study was to identify evidence-based quality-of-care indicators (QCIs) for the management of SAB, and to evaluate the impact of a QCI-based bundle on the management and prognosis of SAB. METHODS A systematic review of the literature to identify QCIs in the management of SAB was performed. Then, the impact of a bundle including selected QCIs was evaluated in a quasi-experimental study in 12 tertiary Spanish hospitals. The main and secondary outcome variables were adherence to QCIs and mortality. Specific structured individualized written recommendations on 6 selected evidence-based QCIs for the management of SAB were provided. RESULTS A total of 287 and 221 patients were included in the preintervention and intervention periods, respectively. After controlling for potential confounders, the intervention was independently associated with improved adherence to follow-up blood cultures (odds ratio [OR], 2.83; 95% confidence interval [CI], 1.78-4.49), early source control (OR, 4.56; 95% CI, 2.12-9.79), early intravenous cloxacillin for methicillin-susceptible isolates (OR, 1.79; 95% CI, 1.15-2.78), and appropriate duration of therapy (OR, 2.13; 95% CI, 1.24-3.64). The intervention was independently associated with a decrease in 14-day and 30-day mortality (OR, 0.47; 95% CI, .26-.85 and OR, 0.56; 95% CI, .34-.93, respectively). CONCLUSIONS A bundle orientated to improving adherence to evidence-based QCIs improved the management of patients with SAB and was associated with reduced mortality.

Mutant Prevention Concentrations of Fluoroquinolones for Enterobacteriaceae Expressing the Plasmid-Carried Quinolone Resistance Determinant qnrA1

ABSTRACT The influence of qnrA1 on the development of quinolone resistance in Enterobacteriaceae was evaluated by using the mutant prevention concentration parameter. The expression of qnrA1 considerably increased the mutant prevention concentration compared to strains without this gene. In the presence of qnrA1, mutations in gyrA and parC genes were easily selected to produce high levels of quinolone resistance.

Qnr-like pentapeptide repeat proteins in Gram-positive bacteria

OBJECTIVES To study the role of Qnr-like pentapeptide repeat proteins (PRPs) from several gram-positive species with quinolone resistance in vitro. METHODS A PCR-based strategy was used to clone and express genes coding for Qnr-like PRPs in Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Clostridium perfringens, C. difficile, Bacillus cereus and B. subtilis in Escherichia coli DH10B. MIC values of nalidixic acid and fluoroquinolones were determined for reference strains and E. coli DH10B harbouring recombinant plasmids containing genes coding for PRPs. RESULTS Amino acid identity of Qnr-like PRPs in gram-positive strains compared with that of the plasmid-mediated quinolone resistance determinants QnrA1, QnrB1 and QnrS1 was in the range of 16% to 22%. Recombinant plasmids coding for Qnr-like PRPs conferred reduced susceptibility to fluoroquinolones (in the range of 0.016 to 0.064 mg/L for ciprofloxacin) and nalidixic acid (from 6 to 12 mg/L), depending on the antimicrobial agent and PRP. The PRP from B. subtilis showed no protective effect. CONCLUSIONS The PRPs analysed conferred a reduced susceptibility phenotype in E. coli; the data provide further evidence of the possible roles in quinolone resistance of PRPs from different gram-positive species. These gram-positive species may constitute a reservoir for Qnr-like quinolone resistance proteins.

In Vitro Effect of qnrA1, qnrB1, and qnrS1 Genes on Fluoroquinolone Activity against Isogenic Escherichia coli Isolates with Mutations in gyrA and parC

ABSTRACT This article provides an analysis of the in vitro effect of qnrA1, qnrB1, and qnrS1 genes, combined with quinolone-resistant Ser83Leu substitutions in GyrA and/or Ser80Arg in ParC, on fluoroquinolone (FQ) resistance in isogenic Escherichia coli strains. The association of Ser83Leu substitution in GyrA, Ser80Arg substitution in ParC, and qnr gene expression increased the MIC of ciprofloxacin to 2 μg/ml. qnr genes present in E. coli that harbored a Ser83Leu substitution in GyrA increased mutant prevention concentration (MPC) values to 8 to 32 μg/ml. qnr gene expression in E. coli may play an important role in selecting for one-step FQ-resistant mutants.

Detection of Plasmid-Mediated Quinolone Resistance Genes in Clinical Isolates of Enterobacter spp. in Spain

ABSTRACT We have studied by PCR and DNA sequencing the presence of the qnrA, qnrB, qnrS, aac(6′)-Ib-cr, qepA, intI1, and ISCR1 genes in 200 clinical isolates of Enterobacter cloacae (n = 153) and E. aerogenes (n = 47) consecutively collected between January 2004 and October 2005 in two hospitals located in Santander (northern Spain) and Seville (southern Spain). Mutations in the quinolone resistance-determining region of gyrA and parC also were investigated in organisms containing plasmid-mediated quinolone resistance genes. The isolates had different resistant phenotypes, including AmpC hyperproduction, extended-spectrum β-lactamase production, resistance or decreased susceptibility to quinolones, and/or resistance to aminoglycosides. Among the 116 E. cloacae isolates from Santander, qnrS1, qnrB5, qnrB2, and aac(6′)-Ib-cr were detected in 22 (19%), 1 (0.9%), 1 (0.9%), and 3 (2.6%) isolates, respectively. Twenty-one, 17, and 2 qnrS1-positive isolates also contained blaLAP-1, intI1, and ISCR1, respectively. A qnrB7-like gene was detected in one E. aerogenes isolate from Santander. No plasmid-mediated quinolone resistance gene was detected in the isolates from Seville. The qnrS1-containing isolates corresponded to four pulsed-field gel electrophoresis patterns and showed various levels of resistance to quinolones. Six isolates were susceptible to nalidixic acid and presented reduced susceptibility to ciprofloxacin. The qnrS1 gene was contained in a conjugative plasmid of ca. 110 kb, and when the plasmid was transferred to recipient strains that did not have a specific mechanism of quinolone resistance, the ciprofloxacin MICs ranged from 0.047 to 0.125 μg/ml.

Long-Term Control of Endemic Hospital-Wide Methicillin-Resistant Staphylococcus aureus (MRSA): The Impact of Targeted Active Surveillance for MRSA in Patients and Healthcare Workers

OBJECTIVE To evaluate the long-term impact of successive interventions on rates of methicillin-resistant Staphylococcus aureus (MRSA) colonization or infection and MRSA bacteremia in an endemic hospital-wide situation. DESIGN Quasi-experimental, interrupted time-series analysis. The impact of the interventions was analyzed by use of segmented regression. Representative MRSA isolates were typed by use of pulsed-field gel electrophoresis. SETTING A 950-bed teaching hospital in Seville, Spain. PATIENTS All patients admitted to the hospital during the period from 1995 through 2008. METHODS Three successive interventions were studied: (1) contact precautions, with no active surveillance for MRSA; (2) targeted active surveillance for MRSA in patients and healthcare workers in specific wards, prioritized according to clinical epidemiology data; and (3) targeted active surveillance for MRSA in patients admitted from other medical centers. RESULTS Neither the preintervention rate of MRSA colonization or infection (0.56 cases per 1,000 patient-days [95% confidence interval {CI}, 0.49-0.62 cases per 1,000 patient-days]) nor the slope for the rate of MRSA colonization or infection changed significantly after the first intervention. The rate decreased significantly to 0.28 cases per 1,000 patient-days (95% CI, 0.17-0.40 cases per 1,000 patient-days) after the second intervention and to 0.07 cases per 1,000 patient-days (95% CI, 0.06-0.08 cases per 1,000 patient-days) after the third intervention, and the rate remained at a similar level for 8 years. The MRSA bacteremia rate decreased by 80%, whereas the rate of bacteremia due to methicillin-susceptible S. aureus did not change. Eighty-three percent of the MRSA isolates identified were clonally related. All MRSA isolates obtained from healthcare workers were clonally related to those recovered from patients who were in their care. CONCLUSION Our data indicate that long-term control of endemic MRSA is feasible in tertiary care centers. The use of targeted active surveillance for MRSA in patients and healthcare workers in specific wards (identified by means of analysis of clinical epidemiology data) and the use of decolonization were key to the success of the program.

Mutational analysis of quinolone resistance in the plasmid-encoded pentapeptide repeat proteins QnrA, QnrB and QnrS

OBJECTIVES Pentapeptide repeat proteins (PRPs) QnrA, QnrB and QnrS confer reduced susceptibility to quinolones. This study presents an in vitro analysis of the genetic evolution of quinolone resistance mediated by changes in the coding sequences and promoter regions of qnrA1, qnrS1 and qnrB1 genes. METHODS A random mutagenesis technique was used to predict the evolutionary potential of these PRPs against nalidixic acid and fluoroquinolones. After comparing the amino acid sequences of these and other PRPs protecting bacteria from quinolone activity, several conserved positions were found. The role of these residues in their effect against quinolones was evaluated by site-directed mutagenesis. RESULTS Three different phenotypes (similar resistance, higher resistance or lower resistance to quinolones) were obtained in the random mutagenesis assays when compared with wild-type phenotypes. Only one mutant increased quinolone resistance: QnrS1 containing D185Y substitution (4-fold for ciprofloxacin). Using site-directed mutagenesis, residues G56, C72, C92, G96, F114, C115, S116, A117 and L159, according to the sequence of QnrA1, were analysed and despite the wide amino acid variability of the PRPs, most conserved residues analysed were critical to QnrA1, QnrB1 and QnrS1. CONCLUSIONS Amino acid sequences of PRPs QnrA1, QnrB1 and QnrS1 could be already optimized for quinolone resistance. One or several changes appear to be insufficient to obtain variants producing fluoroquinolone clinical resistance (MIC > 1 mg/L). Critical residues for quinolone resistance in PRPs were described. Interestingly, different effects were observed for QnrA1, QnrB1 and QnrS1 with the same substitution in several positions.

Eradication of an extensive outbreak in a neonatal unit caused by two sequential Klebsiella pneumoniae clones harbouring related plasmids encoding an extended-spectrum β-lactamase

Outbreaks caused by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae are difficult to control and closure of an affected unit is sometimes necessary. We describe an outbreak of ESBL-producing K. pneumoniae in a 28-bed neonatal unit that began in August 2005 and lasted for seven months. Weekly rectal swabs were taken from all babies admitted throughout the study period. Review of all procedures, contact precautions, thorough environmental cleaning and restriction of cephalosporin use were implemented. ESBL production was investigated according to CLSI recommendations, and characterised by isoelectric focusing and sequencing. Typing of isolates was assessed by pulsed-field gel electrophoresis. Plasmids were also studied. During the outbreak, 32% of 503 admitted babies became colonised and nine babies developed bacteraemia; all the babies recovered. The outbreak was finally terminated in February 2006. Two distinct clones were observed, the first circulating between August and October 2005, and the second between October 2005 and February 2006. Both of these clones carried the non-ESBL SHV-11 and TEM-4 ESBL. Plasmids harbouring TEM-4 from both clones were similar and molecular analysis suggested horizontal dissemination of a single plasmid between the two clones.

Smaqnr, a new chromosome-encoded quinolone resistance determinant in Serratia marcescens

OBJECTIVES A new pentapeptide repeat (PRP) protein, named SmaQnr, from the clinically relevant species Serratia marcescens, which decreased susceptibility to quinolones when expressed in Escherichia coli, is reported herein. METHODS In silico analysis revealed the presence of a gene encoding a Qnr-like protein that shares 80% amino acid identity with QnrB1 in the S. marcescens strain Db11. Fragments carrying the coding region and the upstream non-coding sequences of eight clinical isolates were cloned and expressed in E. coli. MIC values of quinolones were determined. RT-PCR was used to study expression of these genes in their natural host. Southern hybridization was used to explore the presence of the gene in the genus Serratia. RESULTS Recombinant plasmids encoding SmaQnr reduced susceptibility to fluoroquinolones and nalidixic acid in both E. coli ATCC 25922 and DH10B. Sequences upstream of these genes contain a LexA box. Conventional RT-PCR showed transcription of the analysed Smaqnr genes in their natural hosts. Southern blot analysis suggests the presence of similar genes in several species of the genus Serratia. CONCLUSIONS SmaQnr conferred a reduced susceptibility phenotype against fluoroquinolones in E. coli. These data provide evidence of its possible role in quinolone resistance in S. marcescens. This Gram-negative species may constitute a reservoir for qnr-like quinolone resistance genes.