Carl Urban

Nosocomial Outbreak of Klebsiella Infection Resistant to Late-Generation Cephalosporins

Despite frequently reported nosocomial outbreaks of multiple-drug-resistant Klebsiella pneumoniae [1-9], many antibiotics have proved useful against Klebsiella infections during the last two decades. Late-generation cephalosporins became the drugs of choice with usual minimal inhibitory concentrations less than 1 g/mL [10]. These agents are not hydrolyzed by TEM-1 and SHV-1 -lactamases that cause resistance to ampicillin and carbenicillin [11]. Ominously, however, transferable plasmid-mediated resistance to late-generation cephalosporins in Klebsiella has been reported recently from Europe [12-25], the United States [26-28], South America [29], Asia [26], and Africa [30]. Most reports have described sporadic isolates. Several extensive nosocomial outbreaks have occurred in France [15, 16, 23-25] and three lesser episodes in the United States [31-33]. Late in 1988, an outbreak of ceftazidime-resistant Klebsiella pneumoniae (CRKP) began at Booth Memorial Medical Center (now The New York Hospital Medical Center of Queens), a 487-bed, university-affiliated teaching hospital. This report describes an unusual hospital-wide outbreak of CRKP in the United States and its response to enhanced ceftazidime restriction and infection control measures. Antimicrobial susceptibility of CRKP isolates and our approach to therapy of CRKP infections are also addressed. Methods Nosocomial Isolates During routine infection-control surveillance of multi-resistant nosocomial pathogens, a cluster of CRKP isolates was noted in November 1989. Worksheets in the Clinical Microbiology Laboratory were reviewed retrospectively, and the first such isolate was noted to have occurred in October 1988. Subsequently, information on all CRKP isolates was collected retrospectively from November 1989 to October 1988 and prospectively from December 1989 to October 1990. Biochemical identification required a gram-negative, nonmotile bacillus that fermented lactose with gas production, did not produce H2S, was indole negative, and metabolized citrate and/or malonate. Confirmation with AP1-20E (API Analytab Products; Plainview, New York) or Vitek (Vitek Systems, McDonnel-Douglas; Hazelwood, Missouri) was obtained whenever the above biochemical profile was associated with a susceptibility pattern other than the typical ampicillin and carbenicillin resistance of Klebsiella species. Clinical Data Clinical data were obtained from the charts of all patients harboring CRKP from October 1988 through February 1990. Clinical assessment was determined according to the 1988 Centers for Disease Control and Prevention definitions for nosocomial infections [34]. Susceptibility Testing Susceptibility of clinical isolates was tested initially by the Clinical Microbiology Laboratory according to the National Committee for Clinical Laboratory Standards (NCCLS) using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (BBL Microbiology Systems; Cockeysville, Maryland). Ceftazidime resistance was defined as a zone of inhibition less than 15 mm; susceptibility as greater than 17 mm; and intermediate susceptibility as 15 to 17 mm. Strains demonstrating intermediate susceptibility were included with resistant strains for clinical and microbiological analysis. Ceftizoxime was used as a class representative of all other third-generation cephalosporins. Disc diffusion and macrodilution susceptibility tests were performed by the Infectious Disease Research Laboratory on random CRKP isolates obtained after the outbreak was recognized. In these studies, individual third-generation cephalosporins were tested. Broth macrodilution minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) were performed according to NCCLS guidelines [35]. Antimicrobial Agents Antimicrobial agents were obtained as follows: ticarcillin and potassium clavulanate, Beecham Laboratory, Bristol, Tennessee; ceftazidime, Fujisawa SmithKline Corporation, Philadelphia, Pennsylvania; cefuroxime and ceftazidime, Glaxo Pharmaceuticals, Research Triangle Park, North Carolina; cefoxitin, Hoechst Roussel Pharmaceuticals, Inc., Somerville, New Jersey; cefoxitin and imipenem, Merck Sharp & Dohme, West Point, Pennsylvania; cefotetan, Stuart Pharmaceuticals, Wilmington, Delaware; cefmetazole, The Upjohn Company, Kalamazoo, Michigan; Cefepime, Bristol Meyers Company, Syracuse, New York: piperacillin and tazobactam, Lederle Pharmaceuticals, Pearl River, New York: and moxalactam, Eli Lilly & Company, Indianapolis, Indiana. Results Epidemiology From October 1988 to April 1990, 432 isolates of CRKP were recovered from 155 patients, representing 17.3% of all Klebsiella pneumoniae isolates. The peak incidence rate of 72 patients per 1000 average daily census was reached in November 1989 (Figure 1). This represented a prevalence of 25 colonized or infected patients, or 30% of all those from whom Klebsiella pneumoniae had been isolated. Barrier precautions were instituted on colonized and infected patients, and increased restriction of ceftazidime was implemented. Subsequently, the incidence rate of CRKP among medical and surgical patients declined by approximately 60% within the following year. Figure 1. Incidence of ceftazidime-resistant Klebsiella pneumoniae . Klebsiella pneumoniae Patients were colonized or infected with CRKP after a mean of 37.2 days of hospitalization. Ten percent of patients were identified during the first week, 20% during the second week, 22% during the third and fourth weeks, 30% from the fifth to eighth weeks, and 18% after the eighth week of hospitalization. Thus, 70% of patients were identified after the second week of hospitalization. Clinical records of 133 of the 142 patients harboring CRKP through February 1990 were available for analysis. Of these, 52 (39%) met criteria for CRKP infection in 68 separate sites (Table 1). Infections in surgical patients occurred primarily in the surgical intensive care unit. Among medical patients, infections occurred both on general wards and in the intensive care unit. Eighty-four percent of bacteremias and lower respiratory tract infections occurred in both intensive care units, whereas 66% of urinary infections occurred on the wards. Conversely, 73% of CRKP ward infections were urinary, and 62% of intensive care unit CRKP infections were bacteremic or pulmonary. Table 1. Distribution of Ceftazidime-resistant Klebsiella pneumoniae Infections in 68 Sites among 52 Patients The average age of CRKP-infected patients was 70 years. All suffered from various acute and chronic disorders, most of which were rapidly or ultimately fatal. No patient had an absolute neutrophil count less than 1000/mm3. Previous Antibiotic Use and Ceftazidime-resistant Klebsiella pneumoniae Isolation Sufficient records of antibiotic administration were available for 127 of 142 patients identified through February 1990. Before CRKP was isolated, 91 of 127 patients (72%) received more than 7 days of antibiotic therapy. An average of 4.7 antibiotics was administered per patient before CRKP was isolated. Ceftazidime had been used previously in 41% of all patients from whom CRKP was isolated. However, its use preceded CRKP isolation in 76% of those with pneumonia and in 100% of those with bacteremic pneumonia (five patients). Microbiology and Antibiotic Susceptibility Sixty-two CRKP isolates showed intermediate susceptibility by disc diffusion, and 374 isolates showed complete resistance to ceftazidime. Table 2 lists the susceptibilities of all 436 CRKP isolates identified by the Clinical Microbiology Laboratory from October 1988 to April 1990. Nearly 100% of isolates were resistant to mezlocillin, tobramycin, gentamicin, and tetracycline. More than 90% of isolates were susceptible to ciprofloxacin, cefotetan, and ceftizoxime; all isolates were susceptible to imipenem by disc diffusion testing. Susceptibility to cefazolin and cefoxitin was found in 31.9% and 76.8% of CRKP, respectively. Amikacin resistance was present in 53% of CRKP in contrast to 8% of ceftazidime-susceptible Klebsiella pneumoniae. Table 2. Clinical Laboratory Disc Diffusion Susceptibility of 436 Ceftazidime-resistant Klebsiella pneumoniae Isolates to 17 Antibiotics Disc diffusion susceptibility studies were repeated by the Infectious Disease Research Laboratory on 32 randomly selected CRKP isolates (Table 3). Fifteen -lactam agents were tested. Only imipenem and piperacillin-tazobactam yielded inhibitory zones uniformly above the NCCLS breakpoint. Inhibitory zones for all other antibiotics varied widely, with moxalactam, ceftizoxime, cefotetan, cefoxitin, and ceftriaxone inhibiting more than 90% of isolates. Macrodilution assays showed MIC90 of 64 mg/L or more for all antibiotics tested except imipenem, cefotetan, and moxalactam. However, the MBC90 for cefotetan and moxalactam was greater than 64 mg/L compared with 16 mg/L for imipenem (Table 4). Table 3. Disc Diffusion Susceptibility of 32 Ceftazidime-resistant Klebsiella pneumoniae Isolates to 15 Beta-Lactam Antibiotics Table 4. Macrodilution Susceptibility of 18 Ceftazidime-resistant Klebsiella pneumoniae Isolates to 12 Beta-Lactam Antibiotics* Mechanism of Ceftazidime Resistance Studies that will be reported separately have shown several plasmids and at least five different -lactamases in a randomly selected group of nine CRKP isolates. The -lactamase responsible for ceftazidime hydrolysis had a pI of 5.6 and was common to all resistant strains [36]. Enzymatic characteristics were consistent with TEM-10 and the more recently described TEM-26. Both enzymes have been found in limited clusters associated with previous ceftazidime therapy [27, 33]. In each situation, susceptibility to cephamycins, cefotaxime, and ceftriaxone was reported. However, the bactericidal activity of these agents against ceftazidime-resistant isolates was not measured. Therapy and Outcome Therapy and outcome data were available for 43 patients. All 13 patients receivin

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.

Emergence of Carbapenem-Resistant Klebsiella Species Possessing the Class A Carbapenem-Hydrolyzing KPC-2 and Inhibitor-Resistant TEM-30 β-Lactamases in New York City

Nineteen isolates of carbapenem-resistant Klebsiella species were recovered from 7 hospitals in New York City. Most K. pneumoniae belonged to a single ribotype. Nucleotide sequencing identified KPC-2, a carbapenem-hydrolyzing beta -lactamase. In 3 strains, TEM-30, an inhibitor-resistant beta -lactamase, was detected. Carbapenem-resistant Klebsiella species possessing KPC-2 are endemic in New York City. This study documents the identification of an inhibitor-resistant TEM beta -lactamase in the United States.

Clinical and molecular epidemiology of acinetobacter infections sensitive only to polymyxin B and sulbactam

A nosocomial outbreak of infections due to imipenem-resistant Acinetobacter baumannii occurred in a New York hospital after increased use of imipenem for cephalosporin-resistant klebsiella infections. We identified all A baumannii isolates over 12 months, reviewed corresponding patient records, and compared strains with different antibiotic susceptibility patterns by restriction endonuclease analysis. Environmental surveillance cultures were done before and after institution of control measures. 59 patients harboured imipenem-resistant A baumannii, and 18 were infected. Isolates from patients were resistant to all routinely tested antibiotics, including imipenem. Further studies showed susceptibility to polymyxin B and sulbactam. These isolates were identical by restriction endonuclease analysis to A baumannii isolates susceptible to imipenem alone, or to imipenem and amikacin, but differed from broadly susceptible isolates. Surveillance cultures showed hand and environmental colonisation by imipenem-resistant strains. Infection and colonisation were eliminated by intensive infection control measures, and irrigation of wounds with polymyxin B. Increased use of imipenem against cephalosporin-resistant klebsiella may lead to imipenem resistance among other species, particularly acinetobacter. Such resistance appears to derive from a prior multi-resistant clone, in contrast to one which retains susceptibility to several antibiotics.

Considerations in Control and Treatment of Nosocomial Infections Due to Multidrug-Resistant Acinetobacter baumannii

We sought to control infection due to multidrug-resistant Acinetobacter baumannii (MDR-Ab) by identifying isolates as clonally related, leading to enhanced infection-control measures, including cohorting, surveillance, contact precaution, initial therapy with ampicillin/sulbactam and local polymyxin B, and, more recently, therapy with synergistic antibiotic combinations. Class restriction of cephalosporins has been associated with a reduction in cephalosporins-cephamycin-carbapenem resistance among nosocomial Klebsiella isolates. This has been supplemented by restriction of carbapenem use after an initial 24-h period in an effort to reduce the selection of porin-deficient, carbapenem-resistant A. baumannii and Pseudomonas aeruginosa. Evidence is reviewed suggesting that eradication of MDR-Ab nosocomial colonization may prevent subsequent infection. Relatively few standard antibacterial drugs remain active against MDR-Ab. Published clinical results of therapy with these agents are reviewed, and in vitro evidence of synergy between them is presented that suggests that combination therapy should be studied for enhanced clinical activity.

Multiple mechanisms of methicillin resistance and improved methods for detection in clinical isolates of Staphylococcus aureus.

The mec gene of a number of clinical methicillin-resistant Staphylococcus aureus isolates exhibiting a variety of heterogeneous expression modes was selectively inactivated by allelic replacement mutagenesis. While the resistance level of each of the transformants was reduced, the methicillin MIC for these transformants was well above the MIC for susceptible laboratory strains of S. aureus and was similar to the methicillin MIC for many contemporary clinical isolates which did not react with the mec-specific DNA probe but which showed a low or borderline level of resistance to methicillin. A number of those strains had no detectable beta-lactamase, and for about half of the isolates that did carry plasmid-borne beta-lactamase, elimination of the plasmid caused only partial reduction of the methicillin MIC or no reduction at all. The findings suggest that many contemporary strains of staphylococci harbor a combination of at least three distinct beta-lactam resistance mechanisms: (i) the mechanism related to the acquisition of the foreign mec gene and (ii) a beta-lactamase-dependent and (iii) a beta-lactamase-independent mechanism, each one of which can provide a certain degree of resistance against penicillinase-resistant beta-lactam antibiotics. Images

Abrupt Emergence of a Single Dominant Multidrug-Resistant Strain of Escherichia coli

BACKGROUND Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

In Vitro Double and Triple Synergistic Activities of Polymyxin B, Imipenem, and Rifampin against Multidrug-Resistant Acinetobacter baumannii

ABSTRACT Eight unrelated clinical Acinetobacter baumannii isolates resistant to all commonly used antibiotics were subjected to three-dimensional checkerboard microtiter plate dilution and time-kill studies at one-fourth of their MICs of polymyxin B, imipenem, and rifampin. Synergy was demonstrated with combinations of polymyxin B and imipenem, polymyxin B and rifampin, and polymyxin B, imipenem, and rifampin. Double combinations of polymyxin B and imipenem and of polymyxin B and rifampin were bactericidal for seven of eight isolates, and triple combinations were bactericidal for all isolates within 24 h. Future clinical studies using double and triple therapy with these antibacterials may provide an effective option against potentially lethal infection due to multiresistant Acinetobacter baumannii.

Safety of High-Dose Intravenous Daptomycin Treatment: Three-Year Cumulative Experience in a Clinical Program

BACKGROUND There are limited safety data for high-dose and long-term daptomycin treatment (16mg/kg administered for >or=14 days). We present our experience in 61 patients. METHODS We performed a retrospective chart review for all patients treated with daptomycin at New York Hospital Queens (Flushing) from 1 January 2004 through 30 April 2007; patients were identified through a computerized hospital pharmacy database. RESULTS Sixty-one patients (29 male and 32 female patients; mean age, 66.6 years) received a mean dose of 8 mg/kg of daptomycin for a median of 25 days (range, 14-82 days). Twelve patients (with bone and skin and softtissue infections) did not have an identified microbiologic isolate. Gram-positive infections included bloodstream infection with or without infective endocarditis (n = 32), skin and soft-tissue infection (n = 14), bone and joint infection (n = 9), and intra-abdominal infection (n = 5), and unidentified infection (n = 1). Prosthetic devices were removed from 11 of 20 patients. Grade 1 adverse events occurred in 22 patients and did not lead to daptomycin discontinuation. Fifty-eight patients underwent creatine phosphokinase (CPK) analysis (34 patients had paired CPK analyses at the beginning of and during therapy, and 13 patients had random CPK analysis performed during treatment). Three patients had constitutional and/or musculoskeletal symptoms accompanying CPK levels 110 times upper limit of normal (grade 3). All occurred after 24 days of treatment and improved after daptomycin treatment was discontinued. Two of 3 patients were morbidly obese (body mass index grade III). CONCLUSIONS Daptomycin treatment was well tolerated at a mean dose of 8 mg/kg for a median duration of 25 days. The incidence of symptomatic CPK level elevation was within the range reported with lower doses of daptomycin and/or for shorter treatment durations.

Fluoroquinolone-Resistant Streptococcus pneumoniae Associated with Levofloxacin Therapy

Fluoroquinolone-resistant cultures of Streptococcus pneumoniae were isolated from 2 patients who were treated for pneumonia with levofloxacin. Nucleotide sequence analysis of bacterial DNA showed that the isolates contained mutations in both parC (DNA topoisomerase IV) and gyrA (DNA gyrase), which were shown previously to confer fluoroquinolone resistance. With the resistant isolates, the MICs for ciprofloxacin, gatifloxacin, grepafloxacin, levofloxacin, and trovafloxacin were above the maximal serum drug concentrations reported for standard dosage regimens. In contrast, the MICs for gemifloxacin and moxifloxacin were below the maximal serum concentrations. Increased effectiveness at blocking the growth of resistant mutants should make gemifloxacin and moxifloxacin less likely to allow the enrichment of mutants within susceptible populations. Additional resistance mutations in the isolates were readily obtained by plating on gemifloxacin- or moxifloxacin-containing agar. Thus, neither compound is expected to halt further accumulation of resistance mutations once mutant enrichment has been initiated by less potent derivatives.