PurposeCarbapenemases-producing Klebsiella pneumoniae are challenging antimicrobial therapy of hospitalised patients, which is further complicated by colistin resistance. This study describes molecular epidemiological insights into colistin-resistant and carbapenemases-producing clinical K. pneumoniae.Patients and methodsCultures collected from 26 hospitalised patients during 2014–2017 in the main hospital in Molise Region, central Italy, were characterized. The minimum inhibitory concentration for 19 antibiotics was determined, including carbapenems and colistin. Prevalence of resistance-associated genes was investigated through PCR, detecting blaKPC, blaGES, blaVIM, blaIMP, blaNDM, blaOXA-48, blaCTX-M, blaTEM, blaSHV, and mcr-1,2,3,4,5,6,7,8. The mgrB gene was also analysed in colistin-resistant strains by PCR and sequencing assays. K. pneumoniae were typed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).ResultsTwenty out of 26 K. pneumoniae were phenotypically resistant to carbapenems and 19 were resistant to colistin. All isolates harbored blaKPC, and blaSHV, blaTEM and blaVIM were further the most common resistance-associated genes. In colistin-resistant strains, mcr-1,2,3,4,5,6,7,8 variants were not detected, while mutations and insertion elements in mgrB were observed in 68.4% (n=13) in 31.6% (n=6) isolates, respectively. PFGE revealed 12 clusters and 18 pulsotypes at 85% and 95% cut-off, while the Sequence Types ST512 (n=13, 50%), ST101 (n=10, 38.5%), ST307 (n=2, 7.7%) plus a novel ST were detected using MLST.ConclusionAll K. pneumoniae showed a multidrug-resistant phenotype, particularly to carbapenems and colistin. According to national data, blaKPC was the prevailing carbapenemase, followed by blaVIM, while blaTEM and blaSHV were among the most frequent beta-lactamases. Consistent with previous reports in Italy, ST512 was the most common clone, particularly during 2014–15, whilst ST101 became dominant in 2016–17. Colistin resistance was mainly associated with deleterious mutations and transposon in the mgrB gene. Improvements of surveillance, compliance with infection prevention procedures and antimicrobial stewardship are essential to limit the spread of resistant K. pneumoniae.

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Antimicrobial-resistant Klebsiella pneumoniae represent a global public health concern. K. pneumoniae strains isolated during 2010 and 2014-2016 within a single hospital of Molise Region, Central Italy, were analyzed testing antimicrobial susceptibility, clonality by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD)-PCR, and prevalence of carbapenem resistance genes by PCR. Forty isolates (23 wild-type in 2010 and 17 non-wild-type in 2014-2016) were collected from hospitalized patients (65.2 ± 18.1 years old, 75% male, 80% from intensive care unit-ICU). K. pneumoniae showed multidrug-resistant profiles and 15 resistotypes were identified (discriminatory power D = 0.88). The 69.6 and 17.4% of isolates in 2010 resulted intermediate and resistant to imipenem, respectively, and 91.3% was sensitive to meropenem, while 88.2% of isolates of 2014-2016 were resistant to both antibiotics. PFGE identified 16 clusters versus 23 by RAPD, 26 pulsotypes versus 33 RAPD patterns (D ≥ 0.97). PFGE separated strains according to isolation period and identified an outbreak occurred in the ICU during December 2014 and January 2015. No strains harbored bla, bla, bla, and bla genes, as well as AmpC plasmid-mediated beta-lactamases genes. Only K. pneumoniae isolated during 2014-2016 were bla positive. Prevalence of bla was 87 and 76.5% during 2010 and 2014-2016, respectively. No strains colistin-resistant harbored mcr-1 plasmid-mediated resistance gene. The study findings underline an increased circulation of multidrug-resistant K. pneumoniae within the hospital, and the acquisition of carbapenem resistance mechanism. The implementation of surveillance and molecular characterization of isolates are needed to identify outbreaks, reduce the spread of resistance, and guide empirical therapy.

Aims:  In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost‐effective methodological approach based on preliminary PCRs screening was proposed. Methods and Results:  The isolates were analysed by conventional serotyping, multiplex‐PCRs for serogroup and lineage identification and PCR–RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58·10%), II (22·85%), III (12·38%) and IV (6·67%). Among clinical strains, lineage I was more represented (68·75%) than lineage II; whereas, lineage II was more associated with food (90·24%) and environmental (85·72%) isolates. Most of food (89·02%) and environmental (85·71%) isolates were classified into truncated InlA profiles, whereas the 93·75% of clinical strains were associated with a complete form of the protein. Conclusion:  Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs‐based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. Significance and Impact of the Study:  This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost‐effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy.

We estimated the prevalence of Campylobacter spp. in retail meat (n = 352 samples; 104 chicken, 106 pork, and 142 beef) collected in Campobasso, Italy, comparing two microbiological methods. All the isolates were characterized by biomolecular techniques for epidemiological purposes. Campylobacter isolation was performed by selective culture and membrane filtration methods. Phenotypic and genotypic methods for genus and species identification were evaluated together with antimicrobial resistance and plasmid profiling. Sixty-nine (86.2%) samples were positive by selective culture, 49 (61.2%) by membrane filtration, and 38 (47.5%) by both methods. Only 74 of 80 strains were confirmed as Campylobacter spp. by PCR, and two Campylobacter coli were identified as Campylobacter jejuni. Chicken meat was more frequently contaminated than other meats. Selective culture was more sensitive than membrane filtration (85 versus 66%), and specificity of the methods was 98 and 100%, respectively. Among Campylobacter isolates from chicken meat, 86.5% were multidrug resistant. Resistance to ciprofloxacin (51.3%) and enrofloxacin (52.7%) was lower than to nalidixic acid (71.6%). C. coli strains showed the highest cross-resistance for quinolones (82.6%) and fluoroquinolones (60.9%) as well as a high resistance to tetracycline. Plasmids were isolated from six C. coli and two C. jejuni isolates, but no association was detected between antimicrobial resistance and plasmid DNA carriage. Selective culture is considered as the optimal method for Campylobacter isolation, although it was unable to detect all contaminated samples. Membrane filtration provided more specific results but with low sensitivity. A combination of both techniques may offer better results.