Mariano Bernardo

Molecular Epidemiology of Multidrug-Resistant Acinetobacter baumannii in a Tertiary Care Hospital in Naples, Italy, Shows the Emergence of a Novel Epidemic Clone

ABSTRACT The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in two intensive care units of the V. Monaldi university hospital in Naples, Italy, from May 2006 to December 2007. Genotype analysis by pulsed-field gel electrophoresis (PFGE), trilocus sequence-based typing (3LST), and multilocus sequence typing (MLST) of A. baumannii isolates from 71 patients identified two distinct genotypes, one assigned to PFGE group A, 3LST group 1, and ST2 in 14 patients and the other to PFGE group B, 3LST group 6, and ST78 in 71 patients, that we named ST2/A and ST78/B, respectively. Of these, ST2/A corresponded to European clone II identified in the same hospital during 2003 and 2004; ST78/B was a novel genotype that was isolated for the first time in May 2006 but became prevalent during 2007. The ST78/B profile was also identified in five patients from two additional hospitals in Naples during 2007. The ST2/A and ST78/B isolates were resistant to all antimicrobials tested, including carbapenems, but were susceptible to colistin. Both ST2/A and ST78/B isolates possessed a plasmid-borne carbapenem-hydrolyzing oxacillinase gene, bla OXA-58, flanked by ISAba2 and ISAba3 elements at the 5′ and 3′ ends, respectively. The selection of the novel ST78/B A. baumannii clone might have been favored by the acquisition of the bla OXA-58 gene.

High-Dose Daptomycin for Cardiac Implantable Electronic Device–Related Infective Endocarditis

BACKGROUND Cardiac implantable electronic device (CIED)-related endocarditis is a growing challenge because of increasing incidence and significant mortality. Current treatment is based on complete hardware removal coupled with long-term administration of effective and safe antimicrobials. Daptomycin at the dose of 6 mg/kg/day has been found to be effective in staphylococcal endocarditis, but limited data exist on CIED endocarditis. Moreover, whether higher doses could be more effective but equally safe in this setting is currently unknown. METHODS We report here our experience with high-dose daptomycin in the treatment of 25 cases of CIED endocarditis due to staphylococci. RESULTS Patients were mostly elderly and male, with large lead vegetations and severe comorbidities. Pathogens were Staphylococcus epidermidis (56%), Staphylococcus aureus (28%), and other coagulase-negative staphylococci (16%). Only 4 patients (16%) had a normal pretreatment renal function. The median daptomycin daily dose was 8.3 mg/kg (range, 6.4-10.7). Daptomycin was administered for a median of 20 days (range, 8-52). Percutaneous lead extraction was performed in 88% of patients. Two patients (8%) failed to clear bacteremia. The overall clinical success of treatment was 80%, whereas a complete microbiological success was observed in 92% of patients. Creatine phosphokinase values were monitored and increased above normal in 5 cases (20%). No serious adverse event related to high-dose daptomycin was observed and no patient required discontinuation because of muscle toxicity. CONCLUSIONS Our experience suggests that high-dose daptomycin may be a safe therapeutic option in staphylococcal CIED endocarditis and may be associated with high microbiological responses and clinical success.

Molecular epidemiology and mechanisms of rifampicin resistance in Acinetobacter baumannii isolates from Italy

Use of rifampicin (RIF) in combination with colistin (COL) has been proposed for the treatment of multidrug-resistant Acinetobacter baumannii infections owing to in vitro synergism. The aim of the present study was to evaluate the molecular epidemiology and mechanisms of RIF resistance in 57 clinical isolates of A. baumannii in two tertiary care hospitals in Naples (Italy) from 2006 to 2010. Amongst the collection, 36 isolates showed high RIF minimum inhibitory concentrations (MICs) (256 mg/L to ≥512 mg/L), 16 showed intermediate MICs (8-16 mg/L) and 5 had low MICs (4 mg/L). Of the 36 isolates with elevated RIF MICs, 35 were assigned to sequence type ST2 and 1 to ST78. Amongst the 57 isolates, 35 carried at least one mutation in rpoB, including H535L in 9 isolates and double mutations D525N and P544L in 7 isolates, whilst 22 showed no rpoB mutations. Treatment with the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PAβN) of resistant isolates with no mutations in rpoB and different RIF MICs reduced the MIC by >10-fold and restored the synergism between RIF and COL in time-kill studies, whilst it had no effect on strains carrying rpoB mutations. In conclusion, the emergence of elevated RIF MICs in A. baumannii isolates from our geographical area was mostly caused by mutations in rpoB; low to intermediate RIF MICs were also caused by altered membrane permeability to the drug. The phenomenon was contributed by the selection of two prevalent clones both assigned to ST2 genotype. These data may have implications for the correct identification of cases with A. baumannii infection that would not benefit from addition of RIF to COL.

Emergence of colistin resistance without loss of fitness and virulence after prolonged colistin administration in a patient with extensively drug-resistant Acinetobacter baumannii.

The spread of extensively drug-resistant (XDR) gram-negative bacteria has boosted colistin use, with a resultant selection of colistin-resistant, often pandrug-resistant strains. Whether acquisition of further resistance mechanisms translates into a reduced virulence is the subject of active research. In this report, we describe clinical features of an immunocompromised patient who developed infection due to colistin-resistant Acinetobacter baumannii while on long-term colistin therapy. We analyzed phenotypic and genotypic characteristics, molecular mechanisms of colistin resistance, and in vitro and in vivo fitness of sequential colistin-sensitive and colistin-resistant strains isolated from the patient. Both colistin-sensitive and colistin-resistant strains were XDR and showed identical ST78 genotype. At variance with prior reports on colistin-resistant strains of A. baumannii, resistance to colistin due to P233S mutation in PmrB sensor kinase did not associate with any measurable reduction in strain fitness, growth characteristics, and virulence.

Infective endocarditis due to multidrug resistant gram-negative bacilli: Single centre experience over 5 years

BACKGROUND Infective endocarditis (IE) due to gram-negative (GN) bacilli is uncommon. Although multi- and extensively-drug resistant (MDR/XDR) GN infections are emerging, very few data are available on IE due to these microrganisms. METHODS In this study, we describe the clinical characteristics, course and outcome of five contemporary, definite, MDR/XDR GNIE cases seen at our centre. RESULTS All patients had been admitted to a hospital during the 6months before IE onset, 2 were on hemodialysis and 3 on intravenous medications. Three of the 5 cases were hospital-acquired. Intracardiac prosthetic devices were present in all cases (3 central venous lines, 2 prosthetic heart valves, 2 pacemakers). Mean Charlson comorbidity index was 5.8. Causative pathogens were XDR Pseudomonas aeruginosa (2 cases), XDR Acinetobacter baumannii, MDR Burkolderia cepacia and MDR Escherichia coli (1 case each). Concomitant pathogens with a MDR/XDR phenotype were isolated in 4 patients. Both valves and intracardiac devices and left and right sides of the heart were involved. The rate of complications was high. Antibiotic treatment hinged on the use of colistin, a carbapenem or both. Cardiovascular surgical procedures were performed in 3 patients. Despite aggressive therapeutic regimens, outcomes were poor. Clearance of bacteremia was obtained in 3 patients, in-hospital death occurred in 3 patients, only 1 patient survived during follow up. CONCLUSIONS MDR/XDR GN are emerging as a cause of IE in carriers of intracardiac prostheses with extensive healthcare contacts and multiple comorbidities. Resistant GNIE has a complicated course and shows a dismal prognosis.

A Novel IncA/C1 Group Conjugative Plasmid, Encoding VIM-1 Metallo-Beta-Lactamase, Mediates the Acquisition of Carbapenem Resistance in ST104 Klebsiella pneumoniae Isolates from Neonates in the Intensive Care Unit of V. Monaldi Hospital in Naples

The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of β-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim–sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum β-lactamase blaSHV -12 and carbapenem-hydrolyzing metallo-β-lactamase blaV IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with blaV IM-1 into a class I integron, blaSHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2′-phosphotransferase clusters, ant(3″), aph(3″), aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaV IM-1 and blaSHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples.

Increased rate of penicillin non-susceptible strains of N. meningitidis in Naples, Italy

Neisseria meningitidis is a causative agent of community-acquired sepsis and meningitis in humans. These diseases are associated with high mortality and morbidity if treatment is not started promptly. The empiric antibiotic treatment depends on patient factors and local epidemiology. In the past years, an increased number meningococcal strain with reduced susceptibility to penicillin has also been described. We analysed the susceptibility pattern of 11 N. meningitidis strains isolated between 2013 and 2016. Only three (27%) strains were fully susceptible to penicillin, suggesting that a third-generation cephalosporin, instead of penicillin, as empiric therapy is more reliable when an invasive meningococcal disease is suspected.

Treatment of a Klebsiella pneumoniae KPC cellulitis and gut decolonization with ceftazidime/avibactam in a migrant from Libya

KPC-producing Klebsiella pneumoniae (KPC-Kp) is nowadays a global concern. Ceftazidime/avibactam is the most promising novel antibiotic combination available at the moment. We describe the case of a migrant with no risk factors for an infection due to multidrug resistant bacteria. He suffered from a KPC-Kp cellulitis and was treated with a ceftazidime/avibactam-meropenem-fosfomycin combination that not only eradicated the infection but also decontaminated his gut. Ceftazidime/avibactam-based treatment can be useful also in decontamination procedures.

Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency “Ospedale dei Colli,” Naples, Italy

Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency “Ospedale dei Colli,” Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD50 and LD90) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates.

Combining VITEK® 2 with Colistin Agar Dilution Screening Assist Timely Reporting of Colistin Susceptibility

OBJECTIVES The rise in carbapenem resistance among Gram-negative bacteria has renewed interest in colistin. Recently, the EUCAST-CLSI Polymyxin Breakpoints Working Group declared that broth microdilution (BMD) is the only valid method for colistin susceptibility testing. BMD is not easily incorporated into the routine work of clinical laboratories, and usually this test is incorporated serially, resulting in delayed susceptibility reporting. We tested a strategy of combining VITEK® 2 with a 2 μg/mL colistin agar dilution (VITEK® 2/AD) screening plate to improve performance and time to reporting of colistin susceptibility. METHODS Colistin susceptibility for 364 clinical isolates was determined by VITEK® 2/AD and compared with the reference standard BMD according to the ISO 20776-1:2007 and CLSI guidelines. The EUCAST colistin susceptibility breakpoint of ≤2 μg/mL was used. Escherichia coli NCTC 13846 served as quality control strain. Agreement, very major error (VME) and major error rates were determined using ISO 20776-2:2007. RESULTS The VME rate for VITEK® 2 alone was 30.6% (15/49, 95% CI 18.3-45.4%), and was reduced to 10.2% (5/49, 95% CI 3.4-22.2%) using the VITEK® 2/AD combined testing. The combined testing had categorical agreement with BMD of 97% (354/364, 95% CI 95.0-98.7%), and a major error (ME) rate of 1.6% (5/315, 95% CI 0.5-3.7%). Using the combined testing, even against challenging strains, 349 (95.8%, 95% CI 93.3-97.7%) colistin susceptibility results could be reported, and only 15 isolates required further analysis by BMD. DISCUSSION Our method is simple to apply and allows rapid reporting of colistin susceptibility.