Mix and match of KPC-2 encoding plasmids in Enterobacteriaceae-comparative genomics.

We performed comparative sequence analysis of 3 blaKPC-2 encoding plasmids to examine evolution of these plasmids and their dissemination. We found that all of them have an IncN replicon with a newly determined IncN plasmid sequence type (ST), ST15. The 2 Klebsiella pneumoniae (KPN) plasmids also harbor an IncF2A1-B1- replicon. The blaKPC-2 is located in the Tn4401c transposon with a newly discovered mutation in the P2 promoter. Screening of the 27 additional blaKPC-2 carrying plasmids from Enterobacter cloacae, Escherichia coli (EC), and K. pneumoniae showed that: all KPN and EC plasmids are IncN plasmids belonging to ST15; 4/7 KPN and 1/6 EC plasmids contain an additional IncF2A1-B1- replicon; all Enterobacter plasmids belong to neither IncN nor IncF2A1-B1- replicon plasmids; 6/7 KPN and 2/5 EC plasmids carry the mutated P2 promoter. Study of the blaKPC-2 environment, transposon, pMLST, and Inc group suggests transposon and plasmid inter- and intra-species dissemination and evolution.

Development and validation of a multiplex PCR assay for identification of the epidemic ST-258/512 KPC-producing Klebsiella pneumoniae clone.

The Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-KP) sequence type (ST)-258/512 clone is the dominant clone by which KPC has disseminated worldwide. Standard typing methods are time-consuming and are therefore impractical for identification of this clone in the course of an outbreak. Through comparative genomic study, we have previously identified several presumably unique genes of this clone: 1) PILV-like protein (pilv-l), 2) transposase, IS66-family (is-66), and a 3) phage-related protein (prp). Our aims were to 1) test for the presence of these genes using a multiplex PCR in a large, multinational collection of KPC-KP isolates and to 2) validate this assay as a typing method for the identification of the ST-258/512 clone. KPC-KP isolates (n=160) that included both ST-258/512 (group A, n=114) and non-ST-258 (group B, n=46) strains were collected from the following countries: Greece, 20; Israel, 93; Italy, 19; USA, 25; and Colombia, 3. Group B included 30 different STs from various lineages. The pilv-l gene was present in 111/114 of ST-258 isolates, including all of the KPC-negative isolates resulting in a sensitivity of 97%. Using primers for a unique ST-258 pilv-l allele resulted in a specificity of 100%. The sensitivity values of is-66 and prp genes for detecting KPC-KP ST-258 were 83 and 89%, respectively, and the specificity values were 67 and 93%, respectively. PCR for the unique pilv-l ST-258 allele provides a reliable tool for rapid detection of the ST-258 clone. This method can be helpful both in the setting of an outbreak and in a large-scale survey of KPC-KP strains.

Clonal transmission of a rare methicillin-resistant Staphylococcus aureus genotype between horses and staff at a veterinary teaching hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) infection or colonization has become a serious emerging condition in equine hospitals. Following the detection of MRSA in asymptomatic hospitalized horses and in two horses with post-operative wound infections, an investigation was conducted. Twelve of 84 horses (14.3%) and 16 of 139 personnel (11.5%) were MRSA carriers. The profile of the dominant MRSA strain common to horses and staff was multi-drug-resistant, spa-type t535, SCCmec type V, pvl-negative. MLST of a representative isolate yielded sequence type (ST) 5. The risk of MRSA carriage among veterinary personnel was greater in equine veterinarians and full-time technicians in comparison to part-time technicians and to other personnel not working with horses. Strict infection control measures were implemented, horses infected or colonized with MRSA were isolated and decolonization of personnel was attempted. Six months after the intervention, the large animal department personnel and hospitalized horses were all MRSA-negative and the decolonization was considered successful. This outbreak, caused by a rare MRSA strain and involving both hospitalized horses and personnel, further demonstrates the ability of MRSA to spread between animals and humans and emphasizes the importance of infection control measures to decrease the risk for MRSA colonization and infection of both horses and personnel.

High prevalence of methicillin-resistant Staphylococcus aureus among residents and staff of long-term care facilities, involving joint and parallel evolution.

Six long-term care facilities were surveyed for methicillin-resistant Staphylcoccus aureus (MRSA). Among 191 residents, 14% were carriers; 1 strain predominated (ST5-SCCmec II). Among 132 staff members, 11% were positive; 2 strains predominated (ST5-SCCmec II, ST8-SCCmec IV). All strains were Panton-Valentine leukocidin-negative. The epidemiology of MRSA among residents and staff involved joint and parallel evolution.

Complete nucleotide sequence of KPC-3-encoding plasmid pKpQIL in the epidemic Klebsiella pneumoniae sequence type 258.

We have determined the entire DNA sequence of plasmid pKpQIL, the bla(KPC-3)-carrying plasmid harbored by the carbapenem-resistant Klebsiella pneumoniae clone sequence type 258 (ST 258) in Israel. pKpQIL is a 113,637-bp, self-transmissible plasmid that belongs to the incompatibility group IncFII. It consists of a large backbone of a pKPN4-like plasmid and carries the bla(KPC-3)-containing Tn4401a transposon of a pNYC-like plasmid.

Transfer of carbapenem-resistant plasmid from Klebsiella pneumoniae ST258 to Escherichia coli in patient.

Klebsiella pneumoniae carbapenemase (KPC) 3-producing Escherichia coli was isolated from a carrier of KPC-3-producing K. pneumoniae. The KPC-3 plasmid was identical in isolates of both species. The patient's gut flora contained a carbapenem-susceptible E. coli strain isogenic with the KPC-3-producing isolate, which suggests horizontal interspecies plasmid transfer.

Carbapenem-resistant KPC-2-producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008.

All of the carbapenem-resistant Escherichia coli (CREC) isolates identified in our hospital from 2005 to 2008 (n = 10) were studied. CREC isolates were multidrug resistant, all carried bla(KPC-2), and six of them were also extended-spectrum beta-lactamase producers. Pulsed-field gel electrophoresis indicated six genetic clones; within the same clone, similar transferable bla(KPC-2)-containing plasmids were found whereas plasmids differed between clones. Tn4401 elements were identified in all of these plasmids.

Molecular epidemiology, sequence types, and plasmid analyses of KPC-producing Klebsiella pneumoniae strains in Israel.

Sporadic isolates of carbapenem-resistant KPC-2-producing Klebsiella pneumoniae were isolated in Tel Aviv Medical Center during 2005 and 2006, parallel to the emergence of the KPC-3-producing K. pneumoniae sequence type 258 (ST 258). We aimed to study the molecular epidemiology of these isolates and to characterize their bla(KPC)-carrying plasmids and their origin. Ten isolates (8 KPC-2 and 2 KPC-3 producing) were studied. All isolates were extremely drug resistant. They possessed the bla(KPC) gene and varied in their additional beta-lactamase contents. The KPC-2-producing strains belonged to three different sequence types: ST 340 (n = 2), ST 277 (n = 2), and a novel sequence type, ST 376 (n = 4). Among KPC-3-producing strains, a single isolate (ST 327) different from ST 258 was identified, but both strains carried the same plasmid (pKpQIL). The KPC-2-encoding plasmids varied in size (45 to 95 kb) and differed among each of the STs. Two of the Klebsiella bla(KPC-2)-carrying plasmids were identical to plasmids from Escherichia coli, suggesting a common origin of these plasmids. These data indicate that KPC evolution in K. pneumoniae is related to rare events of interspecies spread of bla(KPC-2)-carrying plasmids from E. coli followed by limited clonal spread, whereas KPC-3 carriage in this species is related almost strictly to clonal expansion of ST 258 carrying pKpQIL.

Plasmid pKpQIL encoding KPC-3 and TEM-1 confers carbapenem resistance in an extremely drug-resistant epidemic Klebsiella pneumoniae strain.

OBJECTIVES: An extremely drug-resistant (XDR) clone of KPC-3-producing Klebsiella pneumoniae emerged in Israel in 2006, causing a nationwide outbreak. We aimed to characterize the local KPC-3-encoding plasmid carried by these isolates and study its contribution to antibiotic resistance. METHODS: Mechanisms of carbapenem resistance were investigated in seven selected isolates (isolated between 2006 and 2008) belonging to the epidemic clone. Isolates underwent MIC testing, and were examined for the presence of KPC, Tn4401, class I integron elements and additional antibiotic resistance genes. Plasmids were analysed by transformation, transconjugation, restriction mapping, curing and complementation experiments. Outer membrane protein (OMP) analysis was performed. RESULTS: OMP analysis did not reveal loss of porins. KPC-3-producing K. pneumoniae isolates possessed various plasmids but all harboured a common self-transmissible 105 kb plasmid, termed pKpQIL, encoding bla(TEM-1) and bla(KPC-3). Curing of pKpQIL led to a complete loss of resistance to cephalosporins and carbapenems, proving its crucial role in carbapenem resistance. Transformation of plasmid pKpQIL into the cured Klebsiella strain resulted in full reconstitution of carbapenem resistance. The presence of all Tn4401 transposon elements located upstream of the KPC-3 gene was detected by PCR and sequencing. pKpQIL lacked additional antibiotic resistance genes. CONCLUSIONS: Our findings demonstrate the presence of pKpQIL, a 105 kb KPC-3- and TEM-1-encoding plasmid, in the XDR K. pneumoniae epidemic strain in Israel. pKpQIL is unique and appears consistently in all isolates of this clone over the years. The extensive beta-lactam resistance phenotype of this clone is primarily mediated by this single self-transmissible plasmid.

Detection of aac(6')-Ib-cr in KPC-producing Klebsiella pneumoniae isolates from Tel Aviv, Israel.

OBJECTIVES: We aimed to evaluate the occurrence and characteristics of plasmid-mediated quinolone resistance (PMQR) genes in KPC-producing (KPC-P) Klebsiella pneumoniae (Kpn) isolates in Tel Aviv Medical Center, Israel. METHODS: Forty-seven KPC-P Kpn isolates were studied. Antibiotic susceptibilities were determined by Vitek 2, Etest or agar dilution. Beta-lactamases and PMQR determinants were detected by PCR. For plasmid characterization, transformation, transconjugation, restriction mapping and Southern blot analysis were performed. RESULTS: Six out of 47 (13%) KPC-P isolates carried aac(6')-Ib-cr. Acquisition of aac(6')-Ib-cr-encoding plasmids increased the MIC of ciprofloxacin by 2-fold. In five of six KPC-P isolates, aac(6')-Ib-cr and bla(KPC-2) were encoded on the same plasmid. CONCLUSIONS: The most prevalent PMQR gene in the studied KPC-P K. pneumoniae isolates is aac(6')-Ib-cr. The co-existence of PMQR genes with KPC on the same plasmid poses a serious epidemiological, clinical and public-health threat.

Evaluation of PCR-based testing for surveillance of KPC-producing carbapenem-resistant members of the Enterobacteriaceae family.

The spread of carbapenem-resistant members of the Enterobacteriaceae family (CRE) harboring carbapenemases is an emerging public health threat. As KPC-producing Klebsiella species are endemic in our tertiary care hospital, we aimed to evaluate a PCR-based surveillance test for identification of rectal carriage of KPC-producing CRE. We conducted a surveillance study between May and December 2007. Rectal swabs were collected from patients known to harbor CRE and from contacts of newly discovered patients harboring CRE. Specimens were evaluated by culture and by PCR analysis for blaKPC and were defined as positive if CRE was cultured and blaKPC was identified. Discrepant results between the culture and PCR analysis were resolved by subculturing, repeating the PCR, and performing a hydrolysis assay. Positive CRE cultures prior or subsequent to the time of sampling for the study were also taken into consideration. Sensitivity, specificity, and time to result were calculated. A total of 755 swabs were included. Concordant results were documented for 735 specimens; 51 were positive as determined by both PCR and culture. Discrepancies existed for 20 swabs; 9 were blaKPC negative and CRE culture positive, and 11 were blaKPC positive and CRE culture negative. After repeat testing, a total of 64 samples were classified as blaKPC-positive CRE. The sensitivity and specificity of the PCR analysis were 92.2% and 99.6%, respectively, and those of the culture were 87.5% and 99.4%, respectively. Over the last 3 months of the study, the sensitivity of the PCR improved to 96.3%, versus 77.8% for culture. Time to result was 30 h for the PCR and 60 h (negative) and 75 h (positive) for the CRE culture. blaKPC PCR-based testing is a useful method for the surveillance of KPC-producing CRE. Its main advantage over culturing is a shorter time to result, and it may prove to be more sensitive.

Ertapenem resistance among extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae isolates.

Ertapenem resistance in Klebsiella pneumoniae is rare. We report on an ertapenem-nonsusceptible phenotype among 25 out of 663 (3.77%) extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates in a multicenter Israeli study. These isolates originated from six different hospitals and were multiclonal, belonging to 12 different genetic clones. Repeat testing using Etest and agar dilution confirmed ertapenem nonsusceptibility in only 15/663 (2.3%) of the isolates. The molecular mechanisms of ertapenem resistance in seven single-clone resistant isolates was due to the presence of ESBL genes (CTX-M-2 in four isolates, CTX-M-10 and OXA-4 in one isolate, SHV-12 in one isolate, and SHV-28 in one isolate) combined with the absence of OMPK36. Seven of 10 isolates initially reported as ertapenem nonsusceptible and subsequently classified as susceptible showed an inoculum effect with ertapenem but not with imipenem or meropenem. Population analysis detected the presence of an ertapenem-resistant subpopulation at a frequency of 10(-6). These rare resistant subpopulations carried multiple ESBL genes, including TEM-30, SHV-44, CTX-M-2, and CTX-M-10, and they lacked OMPK36. The clinical and diagnostic significance of the results should be further studied.

Plasmid-mediated qnrB2 and carbapenemase gene bla(KPC-2) carried on the same plasmid in carbapenem-resistant ciprofloxacin-susceptible Enterobacter cloacae isolates.

Fourteen out of 16 carbapenem-resistant quinolone-susceptible Enterobacter cloacae isolates were found to carry qnrB2 and bla(KPC-2) genes encoded on the same plasmid. One isolate also carried the aac(6')-Ib-cr gene. Coexistence of quinolone resistance determinants and bla(KPC-2) on the same plasmid in quinolone-susceptible E. cloacae isolates may have important clinical implications.

Dissemination of the CTX-M-25 family beta-lactamases among Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae and identification of the novel enzyme CTX-M-41 in Proteus mirabilis in Israel.

OBJECTIVES: The CTX-M-25 family of beta-lactamases is a closely related family of enzymes found rarely in the world. We aimed to describe the occurrence and to understand the dissemination of this extended-spectrum beta-lactamase family among Enterobacteriaceae strains in our hospital. METHODS: Fifty-four CTX-M-producing Enterobacteriaceae strains collected from 2000 to 2005 were screened for bla(CTX-M-25) genes by PCR and sequencing. Genetic relatedness was analysed by PFGE. Antibiotic susceptibilities were determined by VITEK-2. Plasmids encoding bla(CTX-M-25)-type genes were isolated, transformed and analysed by Southern blot using a bla(CTX-M-25) probe. Chromosomal location of bla(CTX-M-25)-type was studied by I-CeuI restriction analysis. The bla(CTX-M-25) genetic environment was characterized by PCR mapping and partial sequencing. RESULTS: Ten out of 54 CTX-M-producing isolates (18.5%) carried bla(CTX-M-25) genes, including Klebsiella pneumoniae (n = 4), Escherichia coli (n = 3), Enterobacter cloacae (n = 1) and Proteus mirabilis (n = 2). Isolates were genetically unrelated. Four beta-lactamases were found: CTX-M-25, CTX-M-26, CTX-M-39 and CTX-M-41, a new member of the family (accession no. DQ023162) that differed from CTX-M-25 in three amino acids, Ala80Val, Val106Ile and Ile126Ser. bla(CTX-M-25)-type genes were plasmid-mediated in all genera but P. mirabilis, organized in a class I integron and located downstream of an ISEcp1 element. The genes were encoded on different plasmids with varying degree of similarities. Several antibiotic-resistant determinants conferring resistance to trimethoprim and aminoglycosides existed on the same integron. CONCLUSIONS: bla(CTX-M-25) exists in Israel in different enteric species. Spread of these enzymes within and between species is due to transfer of plasmids with common regions and by dissemination of determinants encoding these genes. CTX-M-41, a novel member of this family, was identified in the chromosome of P. mirabilis.

Molecular and epidemiologic study of polyclonal outbreaks of multidrug-resistant Acinetobacter baumannii infection in an Israeli hospital.

OBJECTIVES: To perform a molecular and epidemiologic investigation of multidrug-resistant (MDR) Acinetobacter baumannii in an institution were polyclonal outbreaks have been observed and determine whether these polyclonal outbreaks had an endogenous origin or were caused by in-hospital patient-to-patient transmission. DESIGN: Retrospective analysis of prospectively collected data. SETTING: An epidemiologic and genotypic investigation of incident cases of MDR A. baumannii infection in an Israeli university tertiary care center. PATIENTS: Hospitalized patients with MDR A. baumannii isolated from clinical samples during a 13-week period, from April 15, 2003, through July 15, 2003. INTERVENTION: All patients with new MDR A. baumannii infections were recruited, and isolates were typed using pulsed-field gel electrophoresis. Data on in-hospital movements and consultations were extracted from computerized databases. Quantification of transmission opportunities (TOPs), defined as encounters between an established carrier and a future carrier of MDR A. baumannii, and analysis of ward clusters were performed. RESULTS: We studied 96 MDR A. baumannii isolates, which belonged to 18 different pulsed-field gel electrophoresis clones. In 65% of cases, TOPs involving patients with the same clone were demonstrated, which is significantly greater than the number of TOPs involving patients with different clones (P=.01). CONCLUSION: Although outbreaks of MDR A. baumannii infection may be polyclonal, we believe that patient-to-patient transmission explains most cases of transmission. Polyclonal local outbreaks reflect several clonal outbreaks occurring simultaneously. The cause of polyclonal outbreaks of A. baumannii infections clustered by ward and time remains to be explained.

Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli hospital.

Carbapenem resistance due to KPC has rarely been observed outside the United States. We noticed a sharp increase in carbapenem-resistant Klebsiella pneumoniae strains possessing KPC in Tel Aviv Medical Center from 2004 to 2006. Sixty percent of the isolates belonged to a single clone susceptible only to gentamicin and colistin and carried the bla(KPC-3) gene, while almost all other clones carried the bla(KPC-2) gene. This rapid dissemination of KPC outside the United States is worrisome.

Plasmid-mediated imipenem-hydrolyzing enzyme KPC-2 among multiple carbapenem-resistant Escherichia coli clones in Israel.

Carbapenem resistance in Escherichia coli is rare. We report four genetically unrelated carbapenem-resistant E. coli isolates cultured from four patients hospitalized in Tel Aviv Medical Center. PCR, sequencing, and Southern blot analysis identified KPC-2 as the imipenem-hydrolyzing enzyme in all four strains, carried on different plasmids with a possible common origin. This is the first discovery of KPC-2 in E. coli and the first report of this enzyme originating outside the United States.

Endocarditis caused by extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae: emergence of resistance to ciprofloxacin and piperacillin-tazobactam during treatment despite initial susceptibility.

Three episodes of bacteremia occurred in the course of prosthetic valve endocarditis caused by an extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae strain. The second isolate developed resistance to ciprofloxacin and the third isolate to piperacillin-tazobactam (PIP-TZ) following sequential therapy with each agent. The first isolate was resistant to PIP-TZ only at high inocula, the second isolate acquired increased transcription of the acrA gene, and the third isolate became resistant to PIP-TZ due to loss of beta-lactamase inhibition by TZ. We question if and how PIP-TZ susceptibility should be reported for ESBL-producing Enterobacteriaceae.

An outbreak of new, nonmultidrug-resistant, methicillin-resistant Staphylococcus aureus strain (sccmec type iiia variant-1) in the neonatal intensive care unit transmitted by a staff member.

A methicillin-resistant Staphylococcus aureus (MRSA) strain was recovered in a neonatal intensive care unit from 3 blood and 3 sputum specimens with antibiotic susceptibility profile characteristic of community-acquired MRSA. Epidemiologic survey resulted in isolation of an identical strain from the nares of one nurse. All isolates carried a new SCCmec type IIIA variant. Treatment of the nurse with topical mupirocin resulted in cessation of the outbreak.