Fabio Arena

CTX-M-type β-lactamases: A successful story of antibiotic resistance

Production of extended-spectrum β-lactamases (ESBLs) is the principal mechanism of resistance to oxyimino-cephalosporins evolved by members of the family Enterobacteriaceae. Among the several ESBLs emerged among clinical pathogens, the CTX-M-type enzymes have proved the most successful in terms of promiscuity and diffusion in different epidemiological settings, where they have largely replaced and outnumbered other types of ESBLs. Originated by the capture and mobilization of chromosomal β-lactamase genes of strains of Kluyvera species, the blaCTX-M genes have become associated with a variety of mobile genetic elements that have mediated rapid and efficient inter-replicon and cell-to-cell dissemination involving highly successful enterobacterial lineages (e.g. Escherichia coli ST131 and ST405, or Klebsiella pneumoniae CC11 and ST147) to yield high-risk multiresistant clones that have spread on a global scale. The CTX-Mβ-lactamase lineage exhibits a striking plasticity, with a large number of allelic variants belonging in several sublineages, which can be associated with functional heterogeneity of clinical relevance. This review article provides an update on CTX-M-type ESBLs, with focus on structural and functional diversity, epidemiology and clinical significance.

In Vivo Emergence of Colistin Resistance in Klebsiella pneumoniae Producing KPC-Type Carbapenemases Mediated by Insertional Inactivation of the PhoQ/PhoP mgrB Regulator

ABSTRACT Colistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniae producing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrB inactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrB deletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrB inactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrB gene, upregulated transcription of phoP, phoQ, and pmrK (which is part of the pmrHFIJKLM operon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniae and were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLM operon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, November 2013 to April 2014.

Consecutive non-replicate clinical isolates (n=191) of carbapenem non-susceptible Enterobacteriaceae were collected from 21 hospital laboratories across Italy from November 2013 to April 2014 as part of the European Survey on Carbapenemase-producing Enterobacteriaceae (EuSCAPE) project. Klebsiella pneumonia carbapenemase-producing K. pneumoniae (KPC-KP) represented 178 (93%) isolates with 76 (43%) respectively resistant to colistin, a key drug for treating carbapenamase-producing Enterobacteriaceae. KPC-KP colistin-resistant isolates were detected in all participating laboratories. This underscores a concerning evolution of colistin resistance in a setting of high KPC-KP endemicity.

MgrB Inactivation Is a Common Mechanism of Colistin Resistance in KPC-Producing Klebsiella pneumoniae of Clinical Origin

ABSTRACT Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrB gene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrB gene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrB gene, including insertions of different types of mobile elements (IS5-like, IS1F-like, or ISKpn14), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrB locus, while the remaining 27 strains (41%) did not show mgrB alterations. Transcriptional upregulation of the phoQ and pmrK genes (part of the phoPQ and pmrHFIJKLM operon, respectively) was observed in all strains with mgrB alterations. Complementation experiments with a wild-type mgrB gene restored colistin susceptibility and basal expression levels of phoQ and pmrK genes in strains carrying different types of mgrB alterations. The present results suggest that mgrB alteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.

mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512.

ABSTRACT A novel mcr variant, named mcr-1.2, encoding a Gln3-to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa).

Large Nosocomial Outbreak of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Traced to Clonal Expansion of an mgrB Deletion Mutant

ABSTRACT We describe a large hospital outbreak (93 bloodstream infections) of colistin-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates which was mirrored by increased colistin consumption. The outbreak was mostly traced to the clonal expansion of an mgrB deletion mutant of an ST512 strain that produced KPC-3.

In Vivo Evolution to Colistin Resistance by PmrB Sensor Kinase Mutation in KPC-Producing Klebsiella pneumoniae Is Associated with Low-Dosage Colistin Treatment

ABSTRACT Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrBL82R in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrBL82R mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

Persistent Carriage and Infection by Multidrug-Resistant Escherichia coli ST405 Producing NDM-1 Carbapenemase: Report on the First Italian Cases

ABSTRACT We report on the first detection of the NDM-1 carbapenemase in Italy, in Escherichia coli isolated in October 2009. Prolonged colonization and relapsing infection by NDM-1-positive E. coli were observed in a patient (index case) with an indirect epidemiological link with areas of endemicity. Transient colonization was apparently observed in another patient linked with the index case.

Oral Gentamicin Gut Decontamination for Prevention of KPC-Producing Klebsiella pneumoniae Infections: Relevance of Concomitant Systemic Antibiotic Therapy

ABSTRACT Gut colonization represents the main source for KPC-producing Klebsiella pneumoniae (KPC-Kp) epidemic dissemination. Oral gentamicin, 80 mg four times daily, was administered to 50 consecutive patients with gut colonization by gentamicin-susceptible KPC-Kp in cases of planned surgery, major medical intervention, or need for patient transfer. The overall decontamination rate was 68% (34/50). The median duration of gentamicin treatment was 9 days (interquartile range, 7 to 15 days) in decontaminated patients compared to 24 days (interquartile range, 20 to 30 days) in those with persistent colonization (P < 0.001). In the six-month period of follow-up, KPC-Kp infections were documented in 5/34 (15%) successfully decontaminated patients compared to 12/16 (73%) persistent carriers (P < 0.001). The decontamination rate was 96% (22/23) in patients receiving oral gentamicin only, compared to 44% (12/27) of those treated with oral gentamicin and concomitant systemic antibiotic therapy (CSAT) (P < 0.001). The multivariate analysis confirmed CSAT and KPC-Kp infection as the variables associated with gut decontamination. In the follow-up period, KPC-Kp infections were documented in 2/23 (9%) of patients treated with oral gentamicin only and in 15/27 (56%) of those also receiving CSAT (P = 0.003). No difference in overall death rate between different groups was documented. Gentamicin-resistant KPC-Kp strains were isolated from stools of 4/16 persistent carriers. Peak gentamicin blood levels were below 1 mg/liter in 12/14 tested patients. Oral gentamicin was shown to be potentially useful for gut decontamination and prevention of infection due to KPC-Kp, especially in patients not receiving CSAT. The risk of emergence of gentamicin-resistant KPC-Kp should be considered.

Epidemic Diffusion of OXA-23-Producing Acinetobacter baumannii Isolates in Italy: Results of the First Cross-Sectional Countrywide Survey

ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAb) is emerging worldwide as a public health problem in various settings. The aim of this study was to investigate the prevalence of CRAb isolates in Italy and to characterize their resistance mechanisms and genetic relatedness. A countrywide cross-sectional survey was carried out at 25 centers in mid-2011. CRAb isolates were reported from all participating centers, with overall proportions of 45.7% and 22.2% among consecutive nonreplicate clinical isolates of A. baumannii from inpatients (n = 508) and outpatients (n = 63), respectively. Most of them were resistant to multiple antibiotics, whereas all remained susceptible to colistin, with MIC50 and MIC90 values of ≤0.5 mg/liter. The genes coding for carbapenemase production were identified by PCR and sequencing. OXA-23 enzymes (found in all centers) were by far the most common carbapenemases (81.7%), followed by OXA-58 oxacillinases (4.5%), which were found in 7 of the 25 centers. In 6 cases, CRAb isolates carried both bla OXA-23-like and bla OXA-58-like genes. A repetitive extragenic palindromic (REP)-PCR technique, multiplex PCRs for group identification, and multilocus sequence typing (MLST) were used to determine the genetic relationships among representative isolates (n = 55). Two different clonal lineages were identified, including a dominant clone of sequence type 2 (ST2) related to the international clone II (sequence group 1 [SG1], SG4, and SG5) and a clone of ST78 (SG6) previously described in Italy. Overall, our results demonstrate that OXA-23 enzymes have become the most prevalent carbapenemases and are now endemic in Italy. In addition, molecular typing profiles showed the presence of international and national clonal lineages in Italy.