Iraida E. Robledo

Detection of KPC in Acinetobacter spp. in Puerto Rico

ABSTRACT During an island-wide PCR-based surveillance study of beta-lactam resistance in multidrug-resistant (MDR) Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus-baumannii complex isolates obtained from 17 different hospitals, 10 KPC-positive Acinetobacter isolates were identified. DNA sequencing of the blaKPC gene identified KPC-2, -3, and -4 and a novel variant, KPC-10. This is the first report of a KPC-type beta-lactamase identified in Acinetobacter species.

Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in Puerto Rico Associated with a Novel Carbapenemase Variant

BACKGROUND Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to almost all antimicrobial agents, and CRKP infections are associated with substantial morbidity and mortality. OBJECTIVE To describe an outbreak of CRKP in Puerto Rico, determine risk factors for CRKP acquisition, and detail the successful measures taken to control the outbreak. DESIGN Two case-control studies. SETTING A 328-bed tertiary care teaching hospital. PATIENTS Twenty-six CRKP case patients identified during the outbreak period of February through September 2008, 26 randomly selected uninfected control patients, and 26 randomly selected control patients with carbapenem-susceptible K. pneumoniae (CSKP) hospitalized during the same period. METHODS We performed active case finding, including retrospective review of the hospitals microbiology database and prospective perirectal surveillance culture sampling in high-risk units. Case patients were compared with each control group while controlling for time at risk. We sequenced the bla(KPC) gene with polymerase chain reaction for 7 outbreak isolates and subtyped these isolates with pulsed-field gel electrophoresis. RESULTS In matched, multivariable analysis, the presence of wounds (hazard ratio, 19.0 [95% confidence interval {CI}, 2.5-142.0]) was associated with CRKP compared with no K. pneumoniae. Transfer between units (adjusted odds ratio [OR], 7.5 [95% CI, 1.8-31.1]), surgery (adjusted OR, 4.0 [95% CI, 1.0-15.7]), and wounds (adjusted OR, 4.9 [95% CI, 1.1-21.8]) were independent risk factors for CRKP compared to CSKP. A novel K. pneumoniae carbapenemase variant (KPC-8) was present in 5 isolates. Implementation of active surveillance for CRKP colonization and cohorting of CRKP patients rapidly controlled the outbreak. CONCLUSIONS Enhanced surveillance for CRKP colonization and intensified infection control measures that include limiting the physical distribution of patients can reduce CRKP transmission during an outbreak.

Surveillance of Carbapenem-Resistant Pseudomonas aeruginosa Isolates from Puerto Rican Medical Center Hospitals: Dissemination of KPC and IMP-18 β-Lactamases

ABSTRACT During a 6-month period, 37/513 (7.2%) Pseudomonas aeruginosa isolates belonging to 13 pulsed-field gel electrophoresis (PFGE) groups from Puerto Rican hospitals were carbapenem nonsusceptible. Seven of 37 isolates from four PFGE groups carried blaIMP-18, and 25/37 isolates from seven PFGE groups carried blaKPC. The results indicated the clonal spread of blaKPC-positive P. aeruginosa isolates into several Puerto Rican hospitals and the dissemination of blaIMP-18 and blaKPC into genetically unrelated isolates.

Detection of the KPC Gene in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii during a PCR-Based Nosocomial Surveillance Study in Puerto Rico

ABSTRACT A 6-month, PCR-based, island-wide hospital surveillance study of beta-lactam resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii was conducted in Puerto Rico. Of 10,507 isolates, 1,239 (12%) unique, multi-beta-lactam-resistant isolates from all geographical regions were identified. The KPC gene was detected in 61 E. coli, 333 K. pneumoniae, 99 P. aeruginosa, and 41 A. baumannii isolates, indicating the widespread dissemination of the KPC gene in clinically significant nosocomial isolates.

Detection of spatial fluctuations of non-point source fecal pollution in coral reef surrounding waters in southwestern Puerto Rico using PCR-based assays.

Human fecal contamination of coral reefs is a major cause of concern. Conventional methods used to monitor microbial water quality cannot be used to discriminate between different fecal pollution sources. Fecal coliforms, enterococci, and human-specific Bacteroides (HF183, HF134), general Bacteroides-Prevotella (GB32), and Clostridium coccoides group (CP) 16S rDNA PCR assays were used to test for the presence of non-point source fecal contamination across the southwestern Puerto Rico shelf. Inshore waters were highly turbid, consistently receiving fecal pollution from variable sources, and showing the highest frequency of positive molecular marker signals. Signals were also detected at offshore waters in compliance with existing microbiological quality regulations. Phylogenetic analysis showed that most isolates were of human fecal origin. The geographic extent of non-point source fecal pollution was large and impacted extensive coral reef systems. This could have deleterious long-term impacts on public health, local fisheries and in tourism potential if not adequately addressed.

Two novel class I integron arrays containing IMP-18 metallo-β-lactamase gene in Pseudomonas aeruginosa clinical isolates from Puerto Rico.

ABSTRACT During a β-lactam resistance surveillance study, 12 IMP-18-positive Pseudomonas aeruginosa isolates belonging to 9 different pulsed-field gel electrophoresis groups were identified. In nine isolates, a class I integron with a novel gene array was identified that contained blaIMP-18 and blaOXA-224, while in two isolates the class I integron contained blaIMP-18 and blaOXA-2 but in a new arrangement. Our findings show the dissemination of two novel class I integrons in P. aeruginosa from different regions of Puerto Rico.

Genetic environment of the KPC gene in Acinetobacter baumannii ST2 clone from Puerto Rico and genomic insights into its drug resistance.

Carbapenems are considered the last-resort antibiotics to treat infections caused by multidrug-resistant Gram-negative bacilli. The Klebsiella pneumoniae carbapenemase (KPC) enzyme hydrolyses β-lactam antibiotics including the carbapenems. KPC has been detected worldwide in Enterobacteriaceae and Pseudomonas aeruginosa isolates associated with transposon Tn4401 commonly located in plasmids. Acinetobacter baumannii has become an important multidrug-resistant nosocomial pathogen. KPC-producing A. baumannii has been reported to date only in Puerto Rico. The objective of this study was to determine the whole genomic sequence of a KPC-producing A. baumannii in order to (i) define its allelic diversity, (ii) identify the location and genetic environment of the blaKPC and (iii) detect additional mechanisms of antimicrobial resistance. Next-generation sequencing, Southern blot, PFGE, multilocus sequence typing and bioinformatics analysis were performed. The organism was assigned to the international ST2 clone. The blaKPC-2 was identified on a novel truncated version of Tn4401e (tentatively named Tn4401h), located in the chromosome within an IncA/C plasmid fragment derived from an Enterobacteriaceae, probably owing to insertion sequence IS26. A chromosomally located truncated Tn1 transposon harbouring a blaTEM-1 was found in a novel genetic environment within an antimicrobial resistance cluster. Additional resistance mechanisms included efflux pumps, non-β-lactam antibiotic inactivating enzymes within and outside a resistance island, two class 1 integrons, In439 and the novel In1252, as well as mutations in the topoisomerase and DNA gyrase genes which confer resistance to quinolones. The presence of the blaKPC in an already globally disseminated A. baumannii ST2 presents a serious threat of further dissemination.

ISEcp1-mediated transposition of blaKPC into the chromosome of a clinical isolate of Acinetobacter baumannii from Puerto Rico

Carbapenems are the last-resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative bacilli. Klebsiella pneumoniae carbapenemase (KPC) hydrolyses β-lactam antibiotics including the carbapenems. KPCs have been detected in Enterobacteriaceae and Pseudomonas aeruginosa isolates worldwide associated with transposon Tn4401 commonly located in plasmids. Acinetobacter baumannii has become an important multidrug-resistant nosocomial pathogen capable of hydrolysing the carbapenem antibiotics. KPC-producing A. baumannii has so far only been reported in Puerto Rico. During a surveillance study, four KPC-producing A. baumannii with identical pulse type were identified in a single institution. The objectives of this study were to characterize the KPC genetic background and the allelic diversity of one of the isolates. Next-generation sequencing and multilocus sequence typing (MLST) were performed. Molecular characterization of the isolate demonstrated blaKPC in Tn4401b located in the bacterial chromosome within a 26.5 kb DNA fragment, which included a KQ-like element (18.9 kb) very similar to that described previously in a K. pneumoniae plasmid and a 7.6 kb DNA fragment with 98 % homology to a putative plasmid from Yersinia pestis strain PY-95. Our data suggested that the 26.5 kb DNA fragment harbouring blaKPC was integrated in the chromosome by a transposition event mediated by the transposase of ISEcp1 found in the KQ-like element. MLST showed a novel sequence type, ST250. To our knowledge, this is the first report of the identification of the genetic background of blaKPC in A. baumannii.

First report of a Pseudomonas aeruginosa clinical isolate co-harbouring KPC-2 and IMP-18 carbapenemases

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KPC Screening by Updated BD Phoenix and Vitek 2 Automated Systems

ABSTRACT Current BD Phoenix and Vitek 2 methodologies were assessed as screens for KPC β-lactamases. Using carbapenem MICs or expert system interpretations as screens, both systems exhibited high (97%) sensitivity in tests with 103 well-characterized Gram-negative isolates, 77 of which were KPC producers.