Characterization of an osteomyelitis case caused by dalbavancin, ceftaroline, and vancomycin non-susceptible methicillin-resistant Staphylococcus aureus.

We report a case of osteomyelitis due to methicillin-resistant Staphylococcus aureus (MRSA) that is also non-susceptible to vancomycin, dalbavancin, ceftaroline, and ceftobiprole, in the absence of exposure to the latter three antibiotics. It was isolated from a patient with a 26-year history of cranial surgeries and episodes of osteomyelitis. Whole-genome sequencing was performed. It was found to belong to ST247 and the mecA gene was detected within the SSCmec type I (1B) gene cassette that lacked the E447K mutation known to produce resistance to ceftobiprole and ceftaroline. However, mutations in other genes related to resistance to these antibiotics were found.

Risk factors for gentamicin-resistant E. coli in children with community-acquired urinary tract infection.

According to many guidelines, gentamicin is the empirical parenteral treatment for children with community-acquired urinary tract infection (CA-UTI). However, increasing resistance rates are reported. The purpose of this study is to analyze risk factors for presenting with a UTI caused by a community-acquired gentamicin-resistant Escherichia coli in children in our hospital and to describe their clinical outcome. A retrospective case-control local study was performed in a tertiary care hospital from January 2014 to December 2016. Cases and controls were children below 14 years old diagnosed in the Emergency Department with febrile CA-UTI caused by gentamicin-resistant and gentamicin-susceptible febrile E. coli strains, respectively. During the study period, 54 cases were included and compared with 98 controls. Patients with chronic conditions were more likely to present with a UTI due to gentamicin-resistant E. coli (OR 3.27; 95% CI 1.37-7.8, p < 0.05), as well as children receiving antibiotic prophylaxis (OR 3.5; 95% CI 1.2-10.1, p < 0.05). Cases had longer hospital stays than controls (5.8 ± 5 days vs. 4.4 ± 4 days, p = 0.017). Gentamicin-resistant strains associated higher rates of cefuroxime (29% vs. 3%), cefotaxime (27% vs. 0%), and quinolone resistance (40.7% vs. 6%) (p < 0.01) and produced more frequently extended-spectrum beta-lactamases (ESBL) (20% vs. 0%, p < 0.01) and carbapenemases (7.4% vs. 0%; p = 0.015). All gentamicin-resistant strains were amikacin-sensitive. The presence of chronic conditions and antibiotic prophylaxis could be potential risk factors for gentamicin-resistant E. coli CA-UTI in children. Simultaneous resistance to cephalosporins, quinolones, and ESBL/carbapenemase production is frequent in these strains.

Risk factors for colonization by carbapenemase-producing enterobacteria at admission to a Surgical ICU: A retrospective study.

INTRODUCTION: In 2011, a hospital-wide outbreak of OXA-48 producing Klebsiella pneumoniae occurred in our hospital, an epidemiological setting of high ESBL-producing K. pneumoniae rates. This study identifies risk factors for colonization with carbapenemase-producing enterobacteria (CPE) at Surgical Intensive Care Unit (SICU) admission. METHODS: A 2-year retrospective study was performed in all patients admitted to the SICU that following routine had a rectal swab collected upon admission. RESULTS: Of 254 patients admitted, 41 (16.1%) harbored CPE (five showing two carbapenemase-producing isolates). Most frequent carbapenemase-producing isolates and carbapenemases were K. pneumoniae (39/46, 84.8%) and OXA-48 (31/46; 76.1%), respectively. Carriers significantly had higher rates of chronic renal disease, previous digestive/biliary endoscopy, hospitalization, ICU/SICU admission, intraabdominal surgery, and antibiotic intake, as well as higher median values of clinical scores (SOFA, SAPS II and APACHE II). In the multivariate analysis (R2=0.309, p<0.001), CPE carriage was associated with prior administration of 3rd-4th generation cephalosporins (OR=27.96, 95%CI=6.88, 113.58, p<0.001), ß-lactam/ß-lactamase inhibitor (OR=11.71, 95%CI=4.51, 30.43, p<0.001), abdominal surgery (OR=6.33, 95%CI=2.12, 18.89, p=0.001), and prior digestive/biliary endoscopy (OR=3.88, 95%CI=1.56, 9.67, p=0.004). CONCLUSIONS: A strong association between production of ESBLs and carriage of CPE (mainly OXA-48 producing K. pneumoniae) was found. According to the model, the co-selection of ß-lactamases by previous exposure to broad-spectrum cephalosporins and ß-lactam/ß-lactamase inhibitors (with lower relative risk), abdominal surgery and prior digestive/biliary endoscopy were factors associated with CPE carriage.

Genomic analysis of the emergence and evolution of multidrug resistance during a Klebsiella pneumoniae outbreak including carbapenem and colistin resistance.

OBJECTIVES: To characterize at the genomic level the evolution of multiresistance during an outbreak of Klebsiella pneumoniae in a burns intensive care unit. The outbreak involved a DHA-1 ß-lactamase-producing strain that later acquired carbapenem and fosfomycin resistance, and in one case colistin resistance. METHODS: The genomes of two isolates were sequenced and compared with a previously sequenced genome. The role of hypermutability was investigated by measuring the mutation frequencies of the isolates and comparison with a collection of control strains. RESULTS: Sequence comparison identified four single-nucleotide variants and two transposon insertions. Analysis of the variants in the whole collection related carbapenem and fosfomycin resistance to a nonsense mutation in the ompK36 porin gene and colistin resistance to an IS1 insertion in the mgrB gene. The plasmid carrying the blaDHA-1 gene was unstable in the absence of antibiotics, and analysis of isolates that had lost the plasmid showed that the porin mutation alone was not sufficient to generate carbapenem resistance. The mutation frequencies were similar among all the strains analysed. CONCLUSIONS: Carbapenem resistance required production of the DHA-1 ß-lactamase and decreased permeability, but fosfomycin resistance depended only on permeability. Resistance to colistin might be related to an alteration in the regulation of the phoPQ system. Hypermutation is not related to the selection of porin mutants. Plasmid instability might be due to the high number of mobile elements and suggests a major role for antibiotic selection pressure in the emergence and evolution of this outbreak.

Mechanisms of linezolid resistance among Staphylococci in a tertiary hospital.

The mechanisms of linezolid resistance among 86 staphylococcal isolates from two intensive care units were investigated. The most frequent was the G2576T mutation in the 23S rRNA (82%). The cfr gene was found in 17% of the isolates, seven S. aureus and eight S. epidermidis isolates. Four of the S. epidermidis isolates had the G2576T mutation and the cfr gene. In four S. haemolyticus isolates, the mechanism could not be identified.

Infections caused by OXA-48-producing Klebsiella pneumoniae in a tertiary hospital in Spain in the setting of a prolonged, hospital-wide outbreak.

OBJECTIVES: We describe clinical and microbiological features of infections caused by OXA-48-producing Klebsiella pneumoniae (O48KP) in the setting of a prolonged, hospital-wide outbreak detected in January 2011. METHODS: Clinical, demographic and microbiological data of patients with growth of O48KP in clinical specimens were collected until December 2011. PCR was used to detect carbapenemase and ß-lactamase genes. The genetic relationships were determined by automated repetitive-sequence-based PCR. RESULTS: Seventy-one patients with clinically guided cultures showing growth of O48KP were identified. Nine were considered to be colonizing rather than causing infection. The most frequent source of infection was the urinary tract (22/62), followed by surgical site infections (17/62). Blood cultures were positive in 23/62 patients. Many patients had significant comorbidity and prolonged hospital stays. In-hospital mortality among patients with O48KP infections was 43.5%. The MIC(90)s of ertapenem, imipenem and meropenem were >32, 16 and 16 mg/L, respectively. No single antimicrobial was active against all the isolates. The antibiotics most active against O48KP were amikacin (97.2% susceptible), colistin (90.1%), tigecycline (73%) and fosfomycin (66.2%). Although eight clones were identified, a predominant clone caused 73.2% of the infections. Multilocus sequence typing (MLST) of the predominant clone gave sequence type (ST) 405 and bla(TEM-1), bla(SHV-76), bla(CTX-M-15) and bla(OXA-1) genes and the insertion sequence IS1999 of the Tn1999 transposon were associated with bla(OXA-48) in this clone. CONCLUSIONS: To our knowledge, this is the largest reported series of infections caused by O48KP in the setting of a single-centre outbreak and provides further input on the clinical relevance of infections caused by O48KP and the difficulties associated with its detection and control.

Genome sequence of OXA-48 carbapenemase-producing Klebsiella pneumoniae KpO3210.

Klebsiella pneumoniae KpO3210 is a OXA-48 carbapenemase-producing isolate obtained from a blood culture in the context of an outbreak in Hospital Universitario La Paz (Madrid, Spain) in 2010. It belongs to the major clone detected during the outbreak and is resistant to all beta-lactams and to several other antibiotics.

Resistance to linezolid in a methicillin-susceptible Staphylococcus aureus clinical isolate without previous exposure to oxazolidinones.

A linezolid-resistant, methicillin-susceptible isolate of Staphylococcus aureus was obtained from an infected surgical wound in an ambulatory patient. The isolate belonged to ST125 and was susceptible to all the antibiotics tested except linezolid (MIC 8 µg/ml) and levofloxacin. Linezolid resistance could be ascribed to the presence of the mutation G2576T in 2 of the 23S rRNA alleles. The mutant alleles were stably maintained in the absence of antibiotic selection.

[Community-acquired methicillin resistant Staphylococcus aureus skin and soft tissue infections in Madrid: prevalence study]. / Prevalencia de infecciones de piel y tejidos blandos producidas por Staphylococcus aureus resistente a Meticilina Comunitario en Madrid.

INTRODUCTION: Since it was first described in the 1990s, Methicillin-Resistant Staphylococcus aureus infection among people with no contact with a hospital setting or with no traditional risk factors, has spread worldwide and is now an important epidemiological and public health problem. METHODS: The present prospective and observational study was carried out from April to November 2010. All adult patients with community-acquired suppurative skin and soft tissue infection (SSTI) attending the Emergency Department were enrolled. Clinical, microbiological and epidemiological features of the infection were assessed. RESULTS: A total of 59 samples were collected from 59 patients and CA-MRSA was isolated in 13 of them. Prevalence of CA-MRSA in patients with suppurative SSTI seen in the emergency department was 22.03%, and was 33.3% in patients with staphylococcal infection. Is worth noting the greater presence of necrosis detected in CA-MRSA lesions. Only 3 patients required hospital admission. Eleven of the 13 strains were Panton-Valentine leucocidin producers, and 5 were resistant to non-betalactam antibiotics. CA MRSA infection is still more frequent in the immigrant population. CONCLUSION: Data on CA-MRSA prevalence in Spain are sparse. This study aims to emphasise the current importance of this emergent pathogen in our area. About one third of suppurative staphylococcal SSTI presenting in our emergency department are caused by this agent, confirming a rapid spread in our country. Some clinical features, such as the high presence of necrosis, are closely related to CA-MRSA.

Meropenem heteroresistance in clinical isolates of OXA-48-producing Klebsiella pneumoniae.

OXA-48-producing Klebsiellapneumoniae isolates often show growth of colonies within inhibition zones in carbapenem diffusion assays. The nature of these colonies was investigated in a series of clinical isolates of OXA-48-producing K. pneumoniae obtained in the context of a hospital outbreak, and they were found to be persistent colonies that reproduced again the same phenotype when they were collected and tested in diffusion assays again. The frequency of mutations conferring resistance to meropenem (8 µg/mL) was determined for the same isolates. The average mutation frequency was 5.47·10-6 (range: 2.59·10-8-5.87·10-5), and the analysis of several resistant mutants showed that all of them had mutations in the ompK36 porin gene. Heteroresistance was investigated using population analysis profiling. The profiles were compatible with mutation frequency assays, and all the colonies analyzed were resistant mutants. In OXA-48-producing K. pneumoniae, the growth of persisters seems to be specific of diffusion assays.

Evaluating the optimal time for amikacin administration with respect to haemodialysis using an in vitro pharmacodynamic simulation against epidemic nosocomial OXA-48 producing Klebsiella pneumoniae ST405 strains.

OBJECTIVES: Bacterial viability and enrichment of resistance resulting from three different amikacin administration schedules with respect to haemodialysis (HD) were assessed against three OXA-48-producing Klebsiella pneumoniae isolated during an outbreak in a Spanish hospital. METHODS: A previously described two-compartment dynamic system was used. Three possible amikacin administration schedules were simulated: (i) haemodialysis immediately after amikacin infusion (pre-HD); (ii) infusion immediately after haemodialysis (post-HD); and (iii) infusion at 50% interdialytic period. Amikacin standard dose (SD) and double dose (DD) were simulated for each schedule. Values of Cmax/MIC, Cmax/MPC (mutant prevention concentration), AUC0-48h/MIC, AUC0-48h/MPC and %TMSW (percentage of time that the concentration was inside the mutant selection window) were determined with experimental data and were correlated with the area under the bacterial killing curve of the total population and the resistant subpopulation. RESULTS: Both with SD and DD, the pre-HD schedule resulted in increases at 48h in bacterial counts of the total population at the expense of enrichment of pre-existing resistant subpopulations from 12h onwards for all strains. The estimated %TMSW that prevented enrichment of resistant mutants was <61.5%. The AUC0-48h/MPC (with values of ≈40 being associated with countering of increases in resistant subpopulations) was better than the %TMSW as a predictive parameter. CONCLUSION: This study showed that the longest times concentrations were above the MPC (i.e. highest AUC0-48h/MPC, lowest %TMSW), the lowest enrichment of resistant subpopulations. This implies use of the highest possible amikacin dose (limited by toxicity) and post-HD as the best administration schedule.

Genomic path to pandrug resistance in a clinical isolate of Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae have spread globally throughout tertiary hospitals. Many Carbapenem-resistant K. pneumoniae clinical isolates are multidrug-resistant (MDR) and may become eventually pandrug-resistant (PDR). Here we present the closed genome of a PDR VIM-1-producing K. pneumoniae strain (KP1050) obtained in a tertiary hospital. The isolate belonged to sequence type 54 (ST54) and had five extrachromosomal elements (four plasmids and a circular phage genome). Most of the antimicrobial resistance genes (ARGs) were located in two clusters borne by two of the plasmids, comprising a class 1 integron that contained up to 14 ARGs including a VIM-1 metallo-ß-lactamase gene, and an IS26 transposon that contained a mobile element from Acinetobacter baumannii encoding the amikacin resistance gene aac(6')-Ian. A MDR isolate obtained 6 years previously was identified (KP1048) retrospectively and was sequenced. Comparison of the two genomes showed that chromosomal mutations in outer membrane porins as well as mutations in the ramR and phoQ genes contributed to increase the resistance spectrum.

Activity of ceftazidime-avibactam against carbapenemase-producing Enterobacteriaceae from urine specimens obtained during the infection-carbapenem resistance evaluation surveillance trial (iCREST) in Spain.

The increasing rates of carbapenemase-producing Enterobacteriaceae (CPE) represent an important threat to health care systems and treatment of CPE infections is a challenge. The aim of the infection-carbapenem resistance evaluation surveillance trial (iCREST) was to determinate the prevalence of CPE in urine specimens in Spain and to evaluate the in vitro activity of ceftazidime-avibactam. Urine specimens (n = 11 826) were included and activity of ceftazidime-avibactam and comparators were investigated by broth microdilution in CPE. Carbapenemases were characterised by polymerase chain reaction (PCR) and sequencing as well as by whole genome sequencing (WGS). Overall prevalence of CPE was 1.6%. OXA-48 was the most prevalent (86.8%), followed by KPC (6.9%), VIM (4.8%), NDM (1.1%) and IMP (0.6%) carbapenemases. Klebsiella pneumoniae was the most common carbapenemase producer (87.8%). An uncommon carbapenemase type (IMP-8) in Spain was identify by WGS in an Enterobacter cloacae isolate, reinforcing the utility of surveillance programmes as effectives tools to detect unexpected genes that encode antimicrobial resistance. Ceftazidime-avibactam showed 100% susceptibility in KPC and OXA-48 producers and the rates of susceptibility in CPE non-susceptible to ceftazidime or meropenem were 92.1% and 96.9%, respectively. Ceftazidime-avibactam could be considered an adequate treatment option for urinary tract infections caused by KPC and OXA-48 producers.

Population structure of OXA-48-producing Klebsiella pneumoniae ST405 isolates during a hospital outbreak characterised by genomic typing.

OBJECTIVES: The aim of this study was to investigate the structure of a broad and sustained hospital outbreak of OXA-48-producing Klebsiella pneumoniae (KpO48) belonging to sequence type 405 (ST405). METHODS: Whole-genome sequencing and comparison of ten ST405 KpO48 isolates obtained from clinical samples in our hospital was performed. Using stringent criteria, 36 single nucleotide polymorphisms (SNPs) were detected (range 0-21 in pairwise comparisons), and allele-specific PCR was used to call the SNPs among a larger set of isolates. RESULTS: Several haplotypes were identified within the population. The haplotypes did not show a spatial structure, but a temporal evolution of sequential haplotype replacements was observed. CONCLUSIONS: The dispersed spatial distribution suggests a reservoir formed by a large pool of colonised patients, and the temporal replacement pattern suggests that the sustained outbreak was composed of several small outbreaks that appeared and rapidly dispersed to several units.

A novel mutation in pmrB mediates colistin resistance during therapy of Acinetobacter baumannii.

Acinetobacter baumannii is a highly versatile nosocomial pathogen. Multidrug resistance among A. baumannii isolates led to the use of colistin, subsequently giving rise to colistin-resistant strains. In this study, the genetic and phenotypic profiles of two colistin-resistant A. baumannii isolates were investigated. Two A. baumannii isolates were obtained from Patient 1 (C071 and C440) and three isolates were obtained from Patient 2 (C080, C314 and C428). Susceptibility profiles were determined by VITEK®2 and Etest. Clonality was determined by RAPD analysis and trilocus multiplex PCR. The pmrCAB operon was sequenced and common carbapenemase genes were screened for by PCR. Doubling times, haemolysis, surface motility, biofilm formation, siderophore production and proteolytic activity were phenotypically determined. Finally, whole-genome sequencing was performed for all five isolates. Isolates C440 and C428 were resistant to colistin and were clonally identical to their sensitive counterparts. The cause of colistin resistance was traced to the previously described P233S mutation in pmrB of C440 and to a novel ΔI19 mutation in pmrB of C428. blaOXA-58-like and blaGES-5 from the strains of Patients 1 and 2, respectively, were also detected. C440 had attenuated proteolytic activity and was positive for siderophore production compared with C071. No difference in in vitro virulence was detected between isolates C080, C314 and C428. In conclusion, one common and one novel mutation were encountered in pmrB from two distinct colistin-resistant A. baumannii isolates. These mutations caused colistin resistance during therapy in two distinct clones, and only one of them had altered in vitro virulence.

Clonality, virulence determinants, and profiles of resistance of clinical Acinetobacter baumannii isolates obtained from a Spanish hospital.

INTRODUCTION: Acinetobacter baumannii is a nosocomial pathogen that is showing increasing rates of carbapenem resistance. Multi-Drug Resistant (MDR) International Clones (ICs), associated with certain oxacillinases, are being reported globally. This organism also harbors numerous virulence determinants. In this study, we aim at characterizing A. baumannii isolated from a Spanish hospital in terms of antimicrobial susceptibility, clonality, carbapenemase genes harbored, and virulence determinants expressed. MATERIALS AND METHODS: Fifty nine clinical bloodstream isolates were obtained from 2009 until 2013. Antimicrobial Susceptibility Testing was performed according to the CLSI guidelines. PFGE and tri-locus PCR typing were then performed in order to determine local and international clonality. PCRs for the detection of common carbapenemases were also performed. Production of hemolysis, biofilms, siderophores, surface motility, and proteolysis were determined phenotypically. Doubling times for selected strains were also calculated. Finally, statistical analysis for detecting associations between these factors was conducted. RESULTS AND DISCUSSION: Carbapenem non-susceptibility was 84.75%, suggesting the immediate need for intervention. PFGE showed the distribution of the majority of the isolates among 7 clusters. Although all three ICs were detected, IC II was predominant at 71.19%. blaOXA-24-like was the most prevalent carbapenemase (62.71%), followed by blaOXA-58-like (13.56%), and blaOXA-23-like (11.86%). Strains pertaining to IC II, and those harboring blaOXA-24-like, were positively associated with α-hemolysis, production of strong biofilms, and siderophore production. Harboring blaOXA-23-like and blaOXA-58-like was associated with attenuated virulence. These associations suggest that an interplay exists between these factors that could be locally exploited. CONCLUSIONS: An alarmingly high rate of carbapenem non-susceptibility has been detected in this study. There was a predominance of IC II and blaOXA-24-like, and those factors were associated with heightened expression of virulence determinants. This association could be exploited for modifying treatment regimens and for improving on infection control protocols in this hospital.