James J. Rahal

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

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.

Efficacy of Interferon α-2b and Ribavirin against West Nile Virus In Vitro

To the Editor: West Nile virus (WNV) infected humans in the Western Hemisphere for the first time in the late summer of 1999. During 1999 and 2000, nine deaths occurred among 80 patients with meningitis or encephalitis in New York City; Westchester County, New York; New Jersey; and Connecticut (1–3). Effective antiviral agents are unknown for infections caused by WNV. Odelola (4) described 83% survival of WNV-infected mice and eradication of virus from brain when 1.5 mg. of ribavirin was administered by intraperitoneal injection after virus inoculation. Survival of controls was 25%. More recently, Jordan et al. have reported inhibition of WNV by a relatively high concentration of ribavirin (200 _M) given after infection of human oligodendroglial cells in vitro (5). Shahar et al. (6) reported protection of fetal mouse spinal cord tissues with mouse alpha and beta interferon before inoculation with WNV. We tested human recombinant interferon alpha-2b and ribavirin for activity against WNV in a primate cell system similar to that used to measure the effect of these agents on Bovine viral diarrhea virus, a cultivatable, closely related surrogate for Hepatitis C virus. Vero cells were cultured at 37o and 5% CO2 in a 96-well microtiter plate. Approximately 13,000 cells were seeded in each well 24 hours before specific concentrations of ribavirin or interferon alpha-2b were added. Approximately 2.5 X 103 PFU of WNV isolated from Culex pipiens (7) was added 1.5-2 hours after or before the addition of interferon alpha-2b or ribavirin to Vero cells. Forty-four hours after treatment, a colorimetric proliferation assay was used to measure viable cells in each treated well according to the protocol of Promega (Madison, WI). Cells exposed to specific concentrations of antiviral compounds, but without WNV, were used as negative controls. Interferon alpha-2b was protective and therapeutic. Interferon alpha-2b inhibited viral cytotoxicity at low dosage when applied before or after infection of cells with WNV. For example, viral protection was observed from 3,000 U/mL to 188 U/mL 2 hours before infection of cells with WNV. Interferon alpha-2b was also therapeutic when applied after cells were infected with WNV. Virus-induced cytotoxicity was inhibited by concentrations of ≥5.9 U/mL when added 1.5 hours after infection (Figure). The optical density 490 values in these tests were significantly different (p<0.05, using Tukey HSD multiple comparison test) when compared with the uninfected cells. Figure Effect of varying concentrations of interferon α-2b (FN) on West Nile virus-infected Vero cells. The vertical axis represents a colorimetric assay of cellular lactic dehydrogenase, which is directly proportional to cell viability and proliferation. ... Ribavirin was protective but not therapeutic in vitro. Cells were protected at dosages of 400 and 500 _M but not at dosages of ≤300 _M of ribavirin applied 2 hours before infection of cells with WNV. A cytotoxic effect of ribavirin occurred at concentrations of 600-1,000 _M. In humans, daily doses of 3 million units of interferon result in serum levels of 10-20 U/mL, well above that required for in vitro efficacy (8). In contrast, oral ribavirin doses of 2,400 mg daily yield a steady-state serum concentration of 3-4 _g/mL after several days, approximately 12-40-fold less than the in vitro inhibitory concentrations of 200-500 _M (50-125 _g/mL) noted by Jordan et al. (5) and in this study. Intravenous administration of 4 g daily, as used in the treatment of Lassa fever, would be required to reach a potentially effective serum concentration (9,10). However, intracellular accumulation and phosphorylation of ribavirin may account for its therapeutic effect in mice (4). We conclude that interferon alpha-2b possesses greater therapeutic activity in vitro than ribavirin, with a potentially greater therapeutic ratio in humans. Whether combination therapy, as employed against hepatitis C, may be optimal requires further study.

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.

Novel antibiotic combinations against infections with almost completely resistant Pseudomonas aeruginosa and Acinetobacter species

For infections with antibiotic-susceptible strains of Pseudomonas aeruginosa, most studies have suggested that combination therapy, usually with a beta -lactam antibiotic plus an aminoglycoside, is preferable for patients with bacteremia and neutropenia. Against infections with Pseudomonas aeruginosa or Acinetobacter baumannii isolates that are resistant to all antibiotics except the polymyxins, several novel antibiotic combinations demonstrate increased activity in vitro compared with that of any single agent. Whether these combinations yield outcomes that are improved over those seen with a polymyxin or other agent alone remains to be determined. However, against infections with species resistant to all antibiotics, including polymyxins, novel combinations are the only remaining therapeutic option.

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.

Evaluation of Vancomycin and Daptomycin Potency Trends (MIC Creep) against Methicillin-Resistant Staphylococcus aureus Isolates Collected in Nine U.S. Medical Centers from 2002 to 2006

ABSTRACT Vancomycin MIC creep has been reported by some institutions but not confirmed in large surveillance studies. We evaluated the possible occurrence of MIC creep when testing vancomycin and daptomycin against methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) by using precise incremental reference MIC methods. Nine hospitals (one in each U.S. census region) randomly selected bloodstream MRSA strains (target, 40/year) from 2002 to 2006. MICs were determined by the reference broth microdilution method using incremental dilutions (eight for each log2 dilution step). Isolates for which vancomycin MICs were >1 μg/ml were typed by pulsed-field gel electrophoresis (PFGE). The vancomycin MIC mode was either 0.625 μg/ml (for eight hospitals) or 0.813 μg/ml (for one hospital), and vancomycin MIC results for 72.9% of strains were between 0.563 and 0.688 μg/ml. No yearly variation in the central tendency of vancomycin MICs for the wild-type population in any medical center was observed; however, when data were analyzed by the geometric mean statistic, vancomycin MIC increases (at three sites) and declines (at three sites) were observed. The daptomycin MIC mode varied from 0.156 μg/ml (2003 to 2005) to 0.219 μg/ml (2002 and 2006), and MIC results for 83.5% (80.3 to 89.2% in each of the centers) of isolates fell between these values. Among PFGE-typed strains, 43 of 55 (78%; from seven hospitals) showed a pattern consistent with that of the USA100 clone, which was represented by all strains from two hospitals and 64 to 88% of strains from five other medical centers; only one strain (2%) was USA300. In conclusion, the perception of MIC creep may vary according to the methods used to analyze the data. Geometric mean MIC data revealed a possible, very-low-level MIC creep at three of nine sites over the 5-year period, which was not evident using modal MICs or the data from all nine hospitals (+0.02 μg/ml). The occurrence of isolates for which the vancomycin MIC was >1 μg/ml was very unusual, with no increased trend, but these organisms were usually clonal (USA100).

Nosocomial Antibiotic Resistance in Multiple Gram-Negative Species: Experience at One Hospital with Squeezing the Resistance Balloon at Multiple Sites

Increased use of antibiotics has led to the isolation of multidrug-resistant bacteria, especially in intensive care units and long-term care facilities. Resistance in specific gram-negative bacteria, including Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, is of great concern, because a growing number of reports have documented mechanisms whereby these microorganisms have become resistant to all available antibacterial agents used in therapy. Reduction in the selection of these multidrug-resistant bacteria can be accomplished by a combination of several strategies. These include having an understanding of the genetics of both innate and acquired characteristics of bacteria; knowing resistance potentials for specific antibacterials; monitoring resistance trends in bacteria designated as problematic organisms within a particular institution on a routine basis; modifying antibiotic formularies when and where needed; creating institutional education programs; and enforcing strict infection-control practices. Strategies appropriate for primary prevention of nosocomial resistance may differ from those required for control of existing epidemic or endemic resistance.