Treatment for klebsiella pneumoniae

Treatment for klebsiella pneumoniae DEFAULT

Klebsiella pneumoniae in Healthcare Settings

General Information

Klebsiella [kleb−see−ell−uh] is a type of Gram-negative bacteria that can cause different types of healthcare-associated infections, including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis. Increasingly, Klebsiella bacteria have developed antimicrobial resistance, most recently to the class of antibiotics known as carbapenems. Klebsiella bacteria are normally found in the human intestines (where they do not cause disease). They are also found in human stool (feces). In healthcare settings, Klebsiella infections commonly occur among sick patients who are receiving treatment for other conditions. Patients whose care requires devices like ventilators (breathing machines) or intravenous (vein) catheters, and patients who are taking long courses of certain antibiotics are most at risk for Klebsiella infections. Healthy people usually do not get Klebsiella infections.

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How Klebsiella bacteria are spread

To get a Klebsiella infection, a person must be exposed to the bacteria. For example, Klebsiella must enter the respiratory (breathing) tract to cause pneumoniae, or the blood to cause a bloodstream infection.

In healthcare settings, Klebsiella bacteria can be spread through person-to-person contact (for example, from patient to patient via the contaminated hands of healthcare personnel, or other persons) or, less commonly, by contamination of the environment. The bacteria are not spread through the air.

Patients in healthcare settings also may be exposed to Klebsiella when they are on ventilators (breathing machines), or have intravenous (vein) catheters or wounds (caused by injury or surgery). Unfortunately, these medical tools and conditions may allow Klebsiella to enter the body and cause infection.

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Preventing Klebsiella from spreading

To prevent spreading Klebsiella infections between patients, healthcare personnel must follow specific infection control precautions (see: Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings ). These precautions may include strict adherence to hand hygiene and wearing gowns and gloves when they enter rooms where patients with Klebsiella–related illnesses are housed. Healthcare facilities also must follow strict cleaning procedures to prevent the spread of Klebsiella.

To prevent the spread of infections, patients also should clean their hands very often, including:

  • Before preparing or eating food
  • Before touching their eyes, nose, or mouth
  • Before and after changing wound dressings or bandages
  • After using the restroom
  • After blowing their nose, coughing, or sneezing
  • After touching hospital surfaces such as bed rails, bedside tables, doorknobs, remote controls, or the phone

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Drug-resistant Klebsiella

Some Klebsiella bacteria have become highly resistant to antibiotics. When bacteria such as Klebsiella pneumoniae produce an enzyme known as a carbapenemase (referred to as KPC-producing organisms), then the class of antibiotics called carbapenems will not work to kill the bacteria and treat the infection. Klebsiella species are examples of Enterobacterales, a normal part of the human gut bacteria, that can become carbapenem-resistant. CRE, which stands for carbapenem-resistant Enterobacterales, are an order of germs that are difficult to treat because they have high levels of resistance to antibiotics. Unfortunately, carbapenem antibiotics often are the last line of defense against Gram-negative infections that are resistant to other antibiotics.

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Treating Klebsiella infections

Klebsiella infections that are not drug-resistant can be treated with antibiotics. Infections caused by KPC-producing bacteria can be difficult to treat because fewer antibiotics are effective against them. In such cases, a microbiology laboratory must run tests to determine which antibiotics will treat the infection.

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What should patients do if they think they have a Klebsiella–related illness?

See a healthcare provider.

What should patients do if they have been diagnosed with a Klebsiella–related illness?

They must follow the treatment regimen prescribed by the healthcare provider. If the healthcare provider prescribes an antibiotic, patients must take it exactly as the healthcare provider instructs. Patients must complete the prescribed course of medication, even if symptoms are gone. If treatment stops too soon, some bacteria may survive and the patient may become re-infected. Patients must wash their hands as often as possible and follow all other hygiene recommendations.

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How would someone know if their Klebsiella infection is drug-resistant?

The healthcare provider will order laboratory tests to determine if the Klebsiella infection is drug-resistant.

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Can a Klebsiella infection spread to the patient’s family members?

If family members are healthy, they are at very low risk of acquiring a Klebsiella infection. It is still necessary to follow all precautions, particularly hand hygiene. Klebsiella bacteria are spread mostly by person-to-person contact and hand hygiene is the best way to prevent the spread of germs.

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Recommendations and Guidelines

For more information about prevention and treatment of HAIs, see the resources below:

Sours: https://www.cdc.gov/hai/organisms/klebsiella/klebsiella.html

Abstract

The prevalence of extended-spectrum β-lactamase (ESBL) production by Klebsiella pneumonia approaches 50% in some countries, with particularly high rates in eastern Europe and Latin America. No randomized trials have ever been performed on treatment of bacteremia due to ESBL-producing organisms; existing data comes only from retrospective, single-institution studies. In a prospective study of consecutive episodes of Klebsiella pneumoniae bacteremia in 12 hospitals in 7 countries, 85 episodes were due to an ESBL—producing organism. Failure to use an antibiotic active against ESBL-producing K. pneumoniae was associated with extremely high mortality. Use of a carbapenem (primarily imipenem) was associated with a significantly lower day mortality than was use of other antibiotics active in vitro. Multivariate analysis including other predictors of mortality showed that use of a carbapenem during the 5-day period after onset of bacteremia due to an ESBL-producing organism was independently associated with lower mortality. Antibiotic choice is particularly important in seriously ill patients with infections due to ESBL-producing K. pneumoniae.

Since their description in the mids, extended-spectrum β-lactamase (ESBL)—producing organisms have become recognized as a worldwide problem [1]. ESBL-producing organisms have been detected in every inhabited continent [2]. Although ESBLs have been detected in a wide variety of gram-negative bacteria, Klebsiella pneumoniae has been found to be the most common species to produce ESBLs [1]. In the United States, between January and June , % of K. pneumoniae isolates from patients in the intensive care unit (ICU) of hospitals participating in the National Nosocomial Infections Surveillance (NNIS) system were nonsusceptible to third-generation cephalosporins or aztreonam [3]. In 10% of NNIS ICUs, at least 27% of all isolates were nonsusceptible to third-generation cephalosporins or aztreonam [3]. (This definition is neither entirely sensitive nor specific for ESBL production but serves as a surrogate marker for production of this type of β-lactamase.)

ESBL-producing organisms may be more common in some parts of Europe, Asia, and South America than in the United States [2, 4, 5]. In a – survey of Klebsiella isolates from ICUs in southern and western Europe, 25% possessed ESBLs [6]. However, in a survey of eastern European centers (e.g., Russia, Poland, and Turkey), almost 50% of K. pneumoniae isolates produced ESBLs [7]. Similarly high rates of ESBL production have been observed in some parts of Asia and Central and South America [5].

Despite the high prevalence of ESBL-producing organisms in many parts of the world, data on the treatment of serious infections due to such organisms remain sparse. To our knowledge, no randomized controlled trials have ever been performed that evaluated the use of various comparator antibiotics in the treatment of serious infections due to ESBL-producing organisms. For a variety of practical reasons, it is unlikely that such a study will ever be performed. In the absence of data from randomized controlled trials, the objective of this report is to describe experience with various agents in the treatment of serious infections due to ESBL-producing organisms. This experience has been derived from the largest prospective study of K. pneumoniae bacteremia ever performed [8, 9].

Patients and Methods

Study design. A prospective, observational study of consecutive, sequentially encountered patients with K. pneumoniae bacteremia was performed in 12 hospitals in South Africa, Taiwan, Australia, Argentina, the United States, Belgium, and Turkey. The study design has been described in detail elsewhere [8]. The study period was 1 January to 31 December Patients aged >6 years with blood cultures positive for K. pneumoniae were enrolled, and a item study form was completed. Patients were observed for 1 month after the first positive blood culture result to assess clinical outcome, including mortality and infectious complications. The study was observational in that administration of antimicrobial agents and performance of other therapeutic management was at the discretion of the patient's physician, not the investigators. The study was approved by institutional review boards, as required by the policies of participating hospitals at the time of the study.

Definitions. Definitions were defined a priori (that is, before data analysis). Nosocomial bacteremia was defined as bacteremia occurring >48 h after admission to the hospital. Severity-of-illness scores included the APACHE III score (for patients in an ICU at the time of onset of bacteremia) [10] and the Pitt bacteremia score [11–13]. The Pitt bacteremia score was calculated using the following criteria: (1) oral temperature: 2 points for a temperature of ⩽35°C or ⩾40°C, 1 point for a temperature of –°C or –°C, and 0 points for a temperature of –°C; (2) hypotension: 2 points for an acute hypotensive event with decreases in systolic and diastolic blood pressure of >30 and >20 mm Hg, respectively, use of intravenous vasopressor agents, or systolic blood pressure <90 mm Hg; (3) receipt of mechanical ventilation: 2 points; (4) cardiac arrest: 4 points; and (5) mental status: alert, 0 points; disoriented, 1 point; stuporous, 2 points; and comatose, 4 points. Immunocompromise was defined as presence of neutropenia or HIV infection, or receipt of prednisone, cyclosporine, or other iatrogenic immunosuppressive agents. Significant underlying disease was defined as a medical history of diabetes mellitus, chronic liver disease, chronic renal failure, HIV infection, malignancy, solid-organ transplantation, or serious burns. Types of infection were determined to be pneumonia, urinary tract infection, meningitis, incisional wound infection, other soft-tissue infections, intraabdominal infection, or primary bloodstream infection, according to Centers for Disease Control and Prevention definitions [14].

Antibiotic therapy for the episode of K. pneumoniae bacteremia comprised agents active in vitro against the blood culture isolate (i.e., the isolate was susceptible to antibiotics, according to NCCLS breakpoints) [15] that were administered for at least 2 days during the 5-day period after the first positive blood culture result. Monotherapy involved administration of only 1 antibiotic with in vitro activity against the infecting isolate for at least 2 days during this period. Combination therapy for the episode of K. pneumoniae bacteremia involved concomitant administration of ⩾2 antibiotics, all of which were active in vitro against the infecting isolate, for at least 2 days during the 5-day period after the first positive blood culture result. Patients who received different active antibiotics nonconcurrently during the 5-day period after the first positive blood culture result were defined as having received sequential monotherapy. Patients who survived for >2 days after the first positive culture result and did not receive any antibiotic with in vitro activity against the blood culture isolate for at least 2 days during the 5-day period after the first positive blood culture result were defined as having received no active antibiotic therapy.

The primary end point of the study was death from any cause within 14 days after the date of the first blood culture positive for K. pneumoniae. Mortality was also assessed as being probably or definitely due to K. pneumoniae bacteremia, the diagnosis of which was made by the patient's physician. This was not used as the primary end point of the study because of the potential for bias in this assessment. A secondary end point was defined as death during the day period after the first blood culture positive for K. pneumoniae. Superinfecting bacteremia was defined as the isolation of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus species, Acinetobacter species, Stenotrophomonas maltophilia, Serratia marcescens, Pseudomonas aeruginosa, Enterobacter species, Citrobacter species, or Candida species from blood cultures during the day period after the initial blood culture positive for K. pneumoniae. Other superinfections were those in which any of the above organisms were isolated from ⩾2 nonblood sites or from the same nonblood site on ⩾2 occasions during the day period after the first positive blood cultures for K. pneumoniae.

Microbiological analysis. ESBL production was phenotypically determined by broth dilution, in accordance with NCCLS performance standards that were current as of January [15]. A ⩾3 two-fold concentration decrease in MICs of cefotaxime—clavulanic acid and ceftazidime—clavulanic acid, compared with MICs when the 2 agents were tested alone, was considered to be phenotypic confirmation of ESBL production. MICs of antibiotics commonly used in the treatment of sepsis due to gram-negative bacteria were determined for the ESBL-producing isolates by the gradient diffusion method (Etest; AB Biodisk).

Statistical analysis. Patient demographic characteristics and laboratory data were entered into the Prophet Statistics computer program, version (AbTech) [16]. The χ2 test or Fisher's exact test were used to compare categorical variables (e.g., the presence or absence of an underlying condition). Continuous variables (e.g., age) were compared using Student's t test or the Mann-Whitney U test. A logistic regression model was used to estimate the effects of multiple factors associated with mortality. The logistic model was developed by entering all variables that had P values ⩽.2 in the univariate analyses into the initial model. Variables were then eliminated one at a time. The model was clustered on the patient to adjust for multiple episodes. The preliminary model was then tested for possible interactions between the main effects. There were no significant interactions. Estimated ORs and 95% CIs were obtained from this model.

Results

A total of episodes of K. pneumoniae bacteremia occurred in patients during the study period; 85 episodes (%) were due to ESBL-producing organisms. A total of 20 (%) of 85 episodes of bacteremia due to ESBL-producing K. pneumoniae resulted in death within 14 days after the first positive blood culture result. Three of these deaths were on the day of or the day after blood samples were cultured. These patients are excluded from analysis of antibiotic use and outcome. Failure to treat with any antibiotic with in vitro activity against isolates recovered during the 5-day period after the positive blood culture result was associated with a significantly higher mortality rate (7 [%] of 11 patients) than was treatment with active antibiotics (10 [%] of 71 patients) (OR, ; 95% CI, –; P = ).

Predictors of mortality associated with ESBL-producingK. pneumoniaebacteremia. For patients who received an antibiotic active in vitro against the ESBL-producing K. pneumoniae strains, factors significantly associated with death due to ESBL-producing K. pneumoniae bacteremia by univariate analysis included accommodation in an ICU at the time of bacteremia (OR, ; 95% CI, –; P = ) and increased severity of illness, as determined by the Pitt bacteremia score (OR, ; 95% CI, –; P = ). Severity of illness, as adjudged by the APACHE III score, was not statistically associated with mortality, most probably because it was only used in 24 patients.

Duration of previous hospitalization, transfer from a nursing home, source of infection, presence of underlying diabetes mellitus, chronic liver disease, renal failure, malignancy, neutropenia, or burns, transplantation, recent surgery, and receipt of corticosteroid therapy were not associated with significantly increased mortality (P > for all factors).

Antibiotic use and outcome associated with ESBL-producingK. pneumoniaebacteremia. Forty-nine episodes of bacteremia due to ESBL-producing K. pneumoniae were treated with monotherapy active in vitro (carbapenems were used in 27 cases; ciprofloxacin was used in 11; cephalosporins were used in 5; β-lactam/β-lactamase inhibitor combinations were used in 4; and amikacin was used in 2), 15 were treated with combination therapy (10 combinations involved carbapenems, and 5 did not involve carbapenems), 7 were treated with sequential monotherapy (5 included a carbapenem), and 11 were treated with antibiotics without in vitro activity against the patient's isolate.

Use of a carbapenem (primarily imipenem) was associated with a significantly lower day mortality due to ESBL-producing K. pneumoniae bacteremia than was use of other active antibiotics. Patients who received a carbapenem as monotherapy or combination therapy in the 5-day period after the first blood culture positive for K. pneumoniae had a significantly lower day all-cause mortality (2 [%] of 42 patients) than did those who received noncarbapenem antibiotics (8 [%] of 29 patients) (OR, ; 95% CI, –; P = ).

There were no statistically significant differences between any of the variables among patients treated with carbapenems or other active antimicrobial agents, except that patients who received carbapenems were more likely to be immunocompromised (table 1). There were also trends toward higher APACHE III scores for patients who received carbapenems, although this difference was not statistically significant.

Table 1

Comparison of variables between patients with Klebsiella pneumoniae bacteremia treated with carbapenems and those treated with other active antibiotics.

Table 1

Comparison of variables between patients with Klebsiella pneumoniae bacteremia treated with carbapenems and those treated with other active antibiotics.

Multivariate analysis was performed using variables (primarily severity-of-illness markers) that were determined to be associated with all-cause death at 14 days after the first positive blood culture result by univariate analysis (table 2). In multivariate analysis, we used the following variables: carbapenem use during the 5-day period after the first blood culture positive for K. pneumoniae, accommodation in an ICU at the time of bacteremia, and severity of illness (as measured by the Pitt bacteremia score). Carbapenem use was independently associated with decreased mortality (OR, ; 95% CI, –; P = ) (table 3). Three additional multivariate analyses, which used variables found on univariate analysis to be associated with day all-cause mortality and and day mortality thought to be due to K. pneumoniae bacteremia, also showed that carbapenem use was independently associated with decreased mortality (table 3).

Table 2

Variables revealed by univariate analysis to be associated with all-cause day mortality among patients with Klebsiella pneumoniae bacteremia.

Table 2

Variables revealed by univariate analysis to be associated with all-cause day mortality among patients with Klebsiella pneumoniae bacteremia.

Table 3

Results of multivariate analyses examining risk factors for mortality associated with bacteremia due to extended-spectrum β-lactamase—producing Klebsiella pneumoniae.

Table 3

Results of multivariate analyses examining risk factors for mortality associated with bacteremia due to extended-spectrum β-lactamase—producing Klebsiella pneumoniae.

Overall, 1 (%) of 27 patients who received carbapenems as the only active antibiotic during the 5-day period after the first positive blood culture result died within 14 days (table 4). The efficacy of carbapenem monotherapy was significantly superior to that of quinolone (mortality, %; OR, ; 95% CI, –; P = ) or noncarbapenem β-lactams (mortality, %; OR, ; 95% CI, –; P = ).

Table 4

Antibiotic choice and mortality associated with bacteremia due to extended-spectrum β-lactamase—producing Klebsiella pneumoniae.

Table 4

Antibiotic choice and mortality associated with bacteremia due to extended-spectrum β-lactamase—producing Klebsiella pneumoniae.

One of 10 patients treated with carbapenems in combination with other active antibiotics (4 patients received amikacin, 4 received ciprofloxacin, and 2 received amikacin-ciprofloxacin) died with 14 days after the positive blood culture result. None of 5 patients treated with other combinations of active therapy died (2 patients received ciprofloxacin-amikacin, 2 received ciprofloxacin-gentamicin, and 1 received ceftazidime-tobramycin). There was no statistically significant advantage associated with receipt of ciprofloxacin in combination with drugs from another antibiotic class (1 of 10 patients who received such combination therapy died), compared with receipt of ciprofloxacin alone (4 of 11 patients who received ciprofloxacin only died) (P = ).

Of the 65 patients who survived for at least 14 days after the onset of ESBL-producing K. pneumoniae bacteremia, 9 (%) of 40 treated with a carbapenem and 4 (%) of 25 treated with a noncarbapenem had a superinfection with a carbapenem-resistant organism (P = ). A total of 5 (%) of 40 patients who received a carbapenem developed superinfectious bacteremia with a multidrug-resistant organism 15–28 days after the first positive blood culture result (MRSA was recovered from 2 patients, carbapenem-resistant P. aeruginosa was recovered from 1, S. maltophilia was recovered from 1, and carbapenem-susceptible Enterobacter cloacae was recovered from 1), compared with 3 (%) of 25 who did not receive a carbapenem (Candida albicans was recovered from 1 patient, MRSA was recovered from 1, and carbapenem-susceptible P. aeruginosa was recovered from 1). An additional 5 patients treated with a carbapenem and 2 patients treated with a noncarbapenem developed superinfections, none of which resulted in bacteremia. These infections involved S. maltophilia and MRSA in 2 and 3 carbapenem-treated patients, respectively, and S. maltophilia and carbapenem-susceptible Acinetobacter baumannii in 1 patient each among those who did not receive a carbapenem.

Discussion

Treatment of serious infection with ESBL-producing K. pneumoniae is difficult because the organisms are frequently resistant to multiple antibiotics. However, in vitro, ESBL-producing organisms may sometimes appear to be susceptible to combination therapy with β-lactams/β-lactamase inhibitors, third- and fourth-generation cephalosporins, aminoglycosides, and quinolones. Susceptibility rates for these antibiotics are 0%–80%, depending on the geographical location of the study site [5–7]. Carbapenems are stable in the presence of hydrolytic effects of ESBLs, which may explain the consistent finding that >98% of ESBL-producing organisms retain susceptibility to either imipenem or meropenem [5–7]. In our study, all ESBL-producing bloodstream isolates were susceptible to imipenem or meropenem, but % were resistant to piperacillin-tazobactam, % were resistant to gentamicin, and % were resistant to ciprofloxacin.

Potentially, the inferior outcome associated with apparently active cephalosporins and β-lactam/β-lactamase inhibitors, compared with that for other antibiotic classes, could be explained by the inoculum effect. This effect (in which MICs of a drug increase up to fold in the presence of increased inocula) is consistently observed with cefotaxime, ceftriaxone, and cefepime against ESBL-producing organisms [17]. An inoculum effect is least frequently observed with carbapenems; piperacillin-tazobactam has an inoculum effect intermediate between those of carbapenems and cephalosporins [17]. In the 2 patients in this series who died after receiving cephalosporin monotherapy, in vitro MICs increased from 8 µg/mL to > µg/mL (for ceftriaxone) and from µg/mL to 8 µg/mL (for cefepime) when a fold increase in the inoculum was used. Animal studies also demonstrate an inoculum effect and adverse outcomes when cephalosporins are used to treat ESBL-producing organisms with MICs of cephalosporin in the susceptible range [18–21]. Apart from the inoculum effect, an alternative explanation for failure of β-lactam antibiotics is failure to achieve pharmacodynamic targets.

Quinolones are not prone to substantially increase their MICs against ESBL-producing strains as the inoculum increases. The relatively poor outcome for patients treated with quinolones in this study is possibly the result of underdosing and subsequent failure to reach pharmacodynamic targets correlated with quinolone efficacy. We can only speculate on this, because we did not record drug doses in this study. Although quinolone resistance is widely believed to be the result of mutations in chromosomal genes coding for targets of quinolone action (gyrA and parC), frequent coexistence of ESBL production and quinolone resistance has been noted [8, 22]. The reasons for this are so far unexplained beyond the potential coexposure of gastrointestinal tract organisms in hospitalized patients to both quinolones and third-generation cephalosporins. Although we found that no patient died who received aminoglycoside monotherapy, because only 2 patients received this therapy, we are therefore reluctant to recommend aminoglycoside monotherapy as a treatment option.

Previous anecdotal reports from single institutions have suggested that carbapenem use may be associated with good clinical outcome in patients with severe infections due to ESBL-producing organisms. In a report from New York City, Meyer et al. [23, p. ] noted that “treatment regimens that included imipenem, in contrast to other antibiotics, yielded the most favorable results,” although they did not provide specific numbers of patients treated. In a report of an outbreak of infection with ESBL-producing organisms in a pediatric hospital, Bingen et al. [24] found that infections in all 17 patients treated with imipenem were cured. In a study of 21 patients infected with ESBL-producing organisms, Burgess et al. [25] found that all patients treated with carbapenems experienced clinical cure; the only treatment failures were observed in patients who had been treated with piperacillin-tazobactam or cefepime, either alone or in combination with a quinolone. In discussions involving 1–4 patients, a variety of other authors have noted favorable outcomes when carbapenems were used to treat serious infections with ESBL-producing organisms [26–32].

Until now, the only data pertaining to treatment of ESBL-producing organisms have been derived from case reports and small retrospective series. Potential deficiencies of such studies are publication bias, the likelihood that outbreaks of oligoclonal infections involve few ESBL types, and the failure to consider factors such as underlying disease severity and source of infection. We have attempted to address some of these limitations by performing a prospective multicountry study of ESBL-producing organisms in consecutive patients with known disease severity and source of infection. Of course, unforeseen bias may occur in any nonrandomized study with a design similar to ours. Optimally, a large, multicenter, randomized, controlled trial should be performed that compares the efficacy of carbapenems with that of other antibiotic classes. However, at the time of writing, there is no apparent industry or governmental support for such a trial. Until such a trial is performed, we recommend carbapenems as the therapy of choice for treating severe infections with ESBL-producing organisms.

acknowledgments

We thank the staff and patients of the following hospitals for their participation in this study: Pittsburgh Veterans Affairs Medical Center (Pittsburgh); Rush–Presbyterian–St. Luke's Medical Center (Chicago); National Cheng Kung University Medical College (Tainan, Taiwan); Royal Brisbane Hospital, Mater Adults Hospital, and Greenslopes Private Hospital (Brisbane, Australia); Hillbrow Hospital and Chris Hani Baragwanath Hospital (Johannesburg, South Africa); Marmara University Hospital (Istanbul, Turkey); University Hospital (Antwerp, Belgium); and San Lucas Hospital and Comunidad Olivos Hospital (Buenos Aires, Argentina).

Financial support. Cottrell Fellowship of the Royal Australasian College of Physicians; Wyeth-Ayerst Pharmaceuticals. Merck and Company provided support for laboratory studies but played no role in study design, conduct of the study, interpretation of the results, or approval of the study prior to publication.

Conflict of interest. K.P.K. has consulted for Abbott Laboratories, AstraZeneca, Bayer, and GlaxoSmithKline; D.L.P. has consulted for Cubist, Wyeth, Elan, Merck, and AstraZeneca; L.B.R. has consulted for Cubist, Wyeth, Elan, Merck, AstraZeneca, Bristol-Myers Squibb, and Ortho-McNeil Pharmaceuticals; K.P.K. has received honoraria from Abbott Laboratories, AstraZeneca, Bayer, and GlaxoSmithKline; D.L.P. has received honoraria from Wyeth, Merck, AstraZeneca, and Pfizer; and L.B.R. has received honoraria from Elan, Wyeth, Merck, and Ortho-McNeil Pharmaceuticals and has given expert testimony on behalf of Wyeth.

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Infection

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Bacteriophage ZCKP1: A Potential Treatment for Klebsiella pneumoniae Isolated From Diabetic Foot Patients

Introduction

Klebsiella pneumoniae belongs to the Enterobacteriaceae family. It primarily affects patients with compromised defenses to cause severe complications. It is a particular problem for patients with diabetes mellitus leading to diabetic foot infections and osteomyelitis (Podschun and Ullmann, ). Once infection is established K. pneumoniae forms a biofilm that enables evasion of the host&#x;s defenses (Akers et al., ; Gupta et al., ). Moreover, phagocytosis by polymorphonuclear granulocytes is dramatically hindered, as K. pneumoniae possesses an outer protective polysaccharide capsule, a key determinant of their subsequent pathogenicity. The capsule suppresses complement components, particularly C3b (Domenico et al., ; Diago-Navarro et al., ). Among many other pathogenicity factors, bone adherence is attributed to adhesin production that may be fimbrial, or non-fimbrial (Malhotra et al., ). Staphylococcus aureus is considered the most frequently implicated bacterium in cases of diabetic foot infection (Richard, ) but recent data indicate that K. pneumoniae is responsible for approximately % of cases (Mukkunnath et al., With rising numbers of diabetes patients and the severity of foot osteomyelitis complications, this represents a considerable economic burden on health providers, notwithstanding the suffering of the individuals affected. In the past, K. pneumoniae was primarily associated with pulmonary and urinary infections, and was only relatively recently recognized as a significant cause of foot osteomyelitis (Dourakis et al., ; Prokesch et al., ).

Foot osteomyelitis is a common and serious problem in diabetic patients resulting chiefly from peripheral neuropathy or, less commonly, by vasculopathy and wound healing impediments (Grayson et al., ). It occurs in approximately two thirds of cases of diabetic foot patients (Grayson et al., ). K. pneumoniae is able to migrate to bone tissues haematogeneously (derived from or transported by blood) or contiguously from areas of local infections in the feet of diabetic patients (Mathews et al., ; Rana et al., ). If not effectively treated, viable cells of the infectious agent can be trapped in the devitalized bone and thus evade host defenses, and eventually cause chronic osteomyelitis (NADE, ; Ross et al., ; Calhoun and Manring, ).

In addition to the virulence characteristics described, the emergence of MDR K. pneumoniae strains, resistant to the last-line antibiotic treatment colistin, is a major concern (Kidd et al., ). Resistance arises from mutations of the mgrB gene, which are stably maintained in Klebsiella populations, from which resistance can be disseminated, in addition to plasmid mediated resistance due to mcr-1 and mcr-2 genes (Cannatelli et al., ). With the advent of the post-antibiotic era, severe cases of osteomyelitis may require more frequent surgical intervention in the form of resection of the infected and necrotic bone (Sanchez et al., ). It is therefore vital to seek alternative therapies to treat K. pneumoniae and other bacterial infections especially in developing countries (Nagel et al., ). Bacteriophage therapy is a good candidate and has been shown, using mice as animal models, to provide significant protection against respiratory and other infections caused by K. pneumoniae such as liver abscesses and bacteremia (Chhibber et al., ; Hung et al., ). Bacteriophage therapy has also been used to treat K. pneumoniae infected burn wound infections, in mice (Malik and Chhibber, ). Intranasal administration of lytic bacteriophage reduced the bacterial burden of K. pneumoniae in the lungs of mice (Cao et al., ). Other studies have characterized a number of diverse lytic bacteriophages to K. pneumoniae belonging to different families and demonstrated their potential in vitro (Bogovazova et al., ; Kesik-Szeloch et al., ; Hoyles et al., ). Bacteriophage therapy is regarded as a simple, safe and highly effective alternative to counter the rising problems associated with multidrug resistant bacteria (Qadir, ; El-Shibiny et al., ). Here we evaluate the lytic activity of bacteriophage ZCKP1 isolated from an environmental freshwater source in Egypt against a MDR K. pneumoniae KP/01 isolated from the foot of a diabetic patient.

Materials and Methods

Bacterial Strains and Growth Media

K. pneumoniae KP/01, used as a host for bacteriophage infection, was isolated from a human clinical diabetic-foot sample from a male patient in May and identified by National Institute of Diabetes using the VITEK method for identification (Cairo, Egypt). Other clinical isolates of K. pneumoniae (n = 21), Proteus mirabilis (n = 18) and E. coli (n = 15) were also isolated by National Institute of Diabetes, for bacteriophage host-range analysis, from wound infection samples and provided to the microbiology research lab at Zewail City. Isolates were kept in tryptone soy broth (TSB; Oxoid, England) containing (w/v) 20% of glycerol, at &#x;80°C. In the following experiments, bacterial strains were grown on tryptic soy agar (TSA; Oxoid, England) overnight, and isolated colonies of bacteria were grown at 37°C, in TSB, to reach OD approximately

Bacterial Identification Using PCR Specific Primers and Gel Electrophoresis

PCR amplification was performed to confirm the identity of the K. pneumoniae isolate (KP/01) using specific primers for 16s RNA gene (forward primer: 5&#x;-ATTTGAAGAGGTTGCAAACGAT-3&#x; and reverse primer: 5&#x;-TTCACTCTGAAGTTTTCTTGTGTTC-3&#x;; Woese and Fox, ; Woese et al., ). Thirty cycles were performed at denaturation temperature of 95°C for 30 s; annealing at 58°C for 60 s and extension at 72°C for 1 min looking for a PCR product of bp length using an Applied Biosystems thermal cycler (Cady et al., ). The PCR product was run on a 1% (w/v) agarose gel to identify its size.

Antibiotic Sensitivity Test

K. pneumoniae KP/01 strain was subjected to antibiotic resistance evaluation against a set of antibiotic discs including: tigecycline (TGC; 15 ¼g), imipenem (IPM; 10 ¼g), piperacillin-tazobactam (TZP; /10 ¼g), levofloxacin (LEV; 5 ¼g), linezolid (LZD; 30 ¼g), ceftazidime (CAZ; 30 ¼g), and cefepime (FEP; 30 ¼g) all from Oxoid (England). Antimicrobial sensitivity testing was performed for strains of K. pneumoniae, E. coli and P. mirabilisby using the disk diffusion methods in accordance with National Committee for Clinical Standards guidelines (Clinical and Laboratory Standards Institute, ). The antibiotics chosen are usually used for the treatment of diabetic foot infections in National Institute of Diabetes, due to their efficacy against members of the Enterobacteriaceae.

Bacteriophage Isolation, Amplification and Purification

Bacteriophages were isolated from environmental water samples from freshwater in El- Maryoteyya-Haram area, Giza, Egypt. K. pneumoniae (KP/01) used as a bacterial host upon which the clear plaquing phage were selected for further characterization. The bacteriophage plaques were purified by repeated single plaque isolation using sterile micropipette tips (Adams, ). All isolated bacteriophages were amplified in liquid culture (TSB) and the lysates were centrifuged at 6, × g for 15 min at 4°C to remove remaining bacterial cells and debris (Marcó et al., ). The supernatant containing phages was then centrifuged for 1 h 15, × g at 4°C. The pellet was resuspended in SM buffer ( mM MgSO4.7 H2O; 10 mM NaCl; 50 mM TrisHCl; pH ) and filtered using ¼m syringe filters (Chromtech, Taiwan). Bacteriophage titers were determined using double-agar overlay plaque assays (Mazzocco et al., ).

Examination of Bacteriophage Morphology by Electron Microscopy

The morphology of bacteriophage ZCKP1 was investigated using transmission electron microscopy at the National Research Center (Cairo, Egypt). Formvar carbon coated copper grids (Pelco International) were immersed into phage suspension, the phage were fixed using glutaraldehyde (% v/v), washed and stained using 2% phosphotungstic acid (pH ). After drying, grids were examined using a transmission electron microscope (JEOL ).

Pulsed Field Gel Electrophoresis (PFGE)

DNA was prepared from bacteriophage ZCKP1 (1010 PFU/ml) to determine the genome size by pulsed field gel electrophoresis (PFGE; Senczek et al., ). Briefly, bacteriophage suspended in agarose plugs were digested with lysis buffer (% w/v SDS [Sigma]; 1% w/v N-Lauryl sarcosine [Sigma]; mM EDTA; 1 mg/ml Proteinase K [Fischer Scientific]), overnight at 55°C. Following washing 2 mm slices of agaraose containing DNA were inserted into the wells of a 1% w/v agarose gel. The gel was run by using a Bio-Rad CHEF DRII system, in X Tris-borate-EDTA, for 18 h at 6 V/cm with a switch time of 30 to 60 s. The size of the genome was determined by comparison to standard concatenated lambda DNA markers (Sigma Aldrich, Gillingham, UK).

Phage DNA Sequencing

Genomic DNA was prepared from phage ZCKP1 (1010 PFU/ml) lysates by proteinase K treatment ( ¼g/ml in 10 mM EDTA pH 8) followed by resin purification using the Wizard DNA kit (Promega, UK) following the manufacturer&#x;s instructions. DNA sequencing was performed using the Illumina MiSeq platform. The data consisted of million paired-end sequence reads of bp in length. Initial processing of the raw data and de novo assembly was performed using CLC Genomics Workbench version (Qiagen, Aarhus, Denmark). ORFs were predicted from PHASTER and manually curated (Arndt et al., ). Nucleotide sequences appear under the GenBank accession number MH

Lytic Profiles of Isolated Bacteriophages

Using double-agar overlay plaque assays (Mazzocco et al., ), the lytic profile of phage ZCKP1 and other isolated phages was determined against a clinical isolate panel when spotted phage concentrations were not 109 PFU/ml [34]. The experiment was performed using log phase bacteria. The panel included bacteria that cause osteomyelitis, including K. pneumoniae, P. mirabilis and E. coli. The lytic activity of bacteriophages was determined based on plaques of clear lysis. If &#x;20 plaques were produced, the tested bacteria were regarded as being sensitive to the phages.

Efficiency of Plating

Bacteriophage ZCKP1 was tested in triplicate over eight decimal dilutions against all the susceptible bacterial strains lysed in the spot assays as previously described (Viazis et al., ). Conditions of these experiments were the same as spot test using log-phase bacteria. Thus, ¼l of all bacterial isolates were added to top agar, and different dilutions of phages were spotted on petri dishes. The plates were incubated overnight at 37°C. Next day, EOP was estimated as the average PFU on target bacteria/average PFU on host bacteria.

Determination of the Frequency of Bacteriophage Insensitive Mutants

The frequency of the emergence of bacteriophage insensitive mutants (BIMs) was estimated as previously described (O&#x;Flynn et al., ). Phage ZCKP1 was mixed with bacterial host strains confirmed to be susceptible to the bacteriophage including strains of K. pneumoniae, P. mirabilis, and E. coli at an MOI of After 10 min of incubation at 37°C, the suspension was serial diluted and spotted using double-agar overlay plaque assays. Plates were incubated overnight and BIM was calculated correspondingly by dividing bacterial viable counts remained after phage infection by initial viable counts. Experiments were conducted in triplicate.

One Step Growth Curve

One step growth curves were performed as previously described (Hyman and Abedon, ). Briefly, KP/01 strain was grown at concentration of 108 and mixed with bacteriophage at multiplicity of infection of 1 and incubated at 37°C for 2 h. Directly after infection and every 10 min, aliquots of ¼l were withdrawn and divided into two volumes of ¼l. Chloroform was added to one of two volumes with a concentration of 1% (v/v); to set intracellular phages free while other ¼l was left with no chloroform addition. After serial dilution, phage titer was estimated by spotting on top agar using double-layer method. Three replicates were conducted for each time interval.

Bacteriophage Potency Against Planktonic Cells

The survival lysis characteristics of phage ZCKP1 were estimated KP/01 in the presence of ZCKP1 phage at multiplicities of infection of , 10 and PFU/CFU was estimated in comparison to bacterial control at a temperature of 37°C (phage-free samples; Armon and Kott, ). Phage infective centers (IC) and plaque forming units (PFU) were also estimated, at different time intervals (0, 5, 10, 20, 30, 40, 60, 90, , and min). IC is the amount of free phage particles released from the bacterial cells, without the need to add chloroform, while PFU refers to the number of nascent phage both inside and outside the bacterial cell. Briefly, two flasks were filled with either bacterial culture at a given concentration (control) or with bacterial culture at the same concentration and bacteriophage matching the desired MOI (Test). At every time interval, the concentration of bacterial control (B), bacterial survival (BS) IC, and PFU were simultaneously estimated. Bacterial concentration were determined using the Miles and Misra method (Miles et al., ), while phage concentration was estimated using double-agar overlay plaque assays by adding chloroform to the aliquot to be estimated in case of PFU determination, or not adding chloroform to calculate the IC.

Bacteriophage ZCKP1 was added to K. pneumoniae KP/01 in log-phase of growth, at 25°C, at an MOI of 1. Bacterial survival, number of infective centers, and number of plaque forming units were estimated periodically at different time intervals (0, 8, 24, 32, and 48 h).

Bacteriophage Activity Against Established Biofilms of K. pneumoniae

The activity of ZCKP1 against established biofilms of KP1/01 was examined using a modification of previously described protocols (Cerca et al., ; Pettit et al., ). One hundred microliter aliquots of K. pneumoniae KP/01 (5 × 106 CFU/ml) in well flat-bottomed polystyrene microtitre plate (Sigma Aldrich) were incubated for 24 h at 37°C. Unattached planktonic cells were carefully removed. The number of bacterial cells in a biofilm per well were estimated to be 107 CFU after 24 h (Mottola et al., ). Using different MOIs (5, 10, and 50), ¼l aliquots of phage ZCKP1 diluted in TSB were added to each well, 1 day after biofilm establishment. Other wells received an equivalent amount of TSB as positive controls. In a parallel experiment, phage was introduced to wells every 4 h carefully replacing the previous suspension (containing TSB, planktonic cells and released phages) without disturbing the established biofilms. The biomass of preformed biofilms was quantified by staining with crystal violet (% w/v). Following washing to remove excess dye with PBS, the crystal violet was solubilized in ethanol (95%). The absorbance was measured using a microplate reader at OD (Biotek, USA). The bacterial counts in biofilms were estimated using an MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide] assay (Serva Electrophores, Germany) as described by Cady et al. (). The absorbance was then measured at nm at 4, 12, and 24 h, using a microplate reader (BioTek, USA). Control and test samples were assayed in triplicate.

Bacteriophage pH and Temperature Stability

The temperature stability of phage ZCKP1 (1010 PFU/ml) was evaluated at 45, 55, 65, 75, 85, and 95°C, at 10 min intervals, over 1 h in adjusted water bath incubator. Immediately after incubation, serial dilutions of phage were spotted in triplicate, using standard double layer technique; on a lawn of host strain (KP/01) to estimate phage titers as previously described (Capra et al., ; Hammerl et al., ).

The bacterial counts of ZCKP1 at different pH values (5, 6, 7, 8, and 9) was determined after 1 h incubation, followed by determining the phage titer as previously described (Hammerl et al., ). Different pH values were achieved in SM phage buffer to maintain comparative conditions.

Statistical Analysis

In all data sets, test and control sets were compared using Student&#x;s t-test. A significance level of was applied in all cases. Analytical statistics were undertaken using GraphPad PRISM version for Windows (GraphPad Software, La Jolla, USA).

Results

Klebsiella Identification and Sensitivity to Antibiotics

The identity of the KP/01 strain was confirmed to be K. pneumoniae by PCR, by the presence of bp band corresponding to conserved region in 16s RNA gene of K. pneumoniae, following amplification with the specific primers. The antibiotic sensitivity of K. pneumoniae isolate KP/01 was tested using the disc diffusion method and the results showed that K. pneumoniae isolate KP/01 was sensitive to tigecycline (TGC), imipenem (IPM) and piperacillin-tazobactam (TZP) but resistant to levofloxacin (LEV), linezolid (LZD), ceftazidime (CAZ) and cefepime (FEP).

Bacteriophage Isolation

Bacteriophages were isolated from freshwater near the pyramids of Egypt in Giza. Selection of the bacteriophage was undertaken upon serial passage according to their ability to lyse a broad range of K. pneumoniae isolates and other pathogens causing osteomyelitis, generate reproducible clear zones of lysis, produce hallow zones around lysis zones indicative of exopolysaccharide depolymerase activity and capable of replication to produce high titers on the selected host with respect to time. Bacteriophage ZCKP1 fulfilled these criteria.

Morphology of Lytic ZCKP1 Phage

Electron microscopy revealed that ZCKP1 had an icosahedral head and contractile tail with collar, and base plate, and therefore typical of phages belonging to the family of Myoviridae (Figure 1). The proportions of the phage head and tail length were also typical of the Myoviridae with the head size being 80 ± nm while tail length was calculated to be ± nm.

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Figure 1. Transmission electronmicroscopic image of phage ZCKP1.

Phage Genome

Bacteriophage ZCKP1 contains a double-stranded DNA genome estimated to be kbp by PFGE, which is comparable to values indicated by International Committee on Taxonomy of Viruses (ICTV) for bacteriophages belonging to the Myoviridae family. DNA sequencing of the phage DNA enabled de novo assembly and accurate size determination of a circular permuted genome of , bp with a G + C content of %. The genome contained open reading frames, the majority of which are hypothetical proteins or recognized in BLASTP database searches as phage proteins without any ascribed function. Reading frames for which putative functional information could be ascribed to the products appear in Supplementary Table 1. Notably these include the phage structural proteins, nucleotide metabolism and components of the replication machinery that are conserved amongst Myoviridae infecting hosts within the Enterobacteriaceae. Of interest are enzymes that have the potential to modify infected cell surface polysaccharides that may impede superinfection. These include an O-antigen biosynthesis protein, a glycosyltransferase and a wcaM superfamily protein associated with colonic acid biosynthesis clusters present in Enterobacteriaceae that feature exopolysaccharide production. Four genes encoding proteins related to tellurite resistance are present. Tellurite resistance is frequently used for selection in culture isolation media but is not used for antimicrobial therapy. The genes are thought to contribute to colicin and phage resistance (Taylor and Summers, ), which may provide reasons for their presence in phage ZCKP1 in that colicin resistance will provide a selective advantage to the phage infected cell and phage resistance to prevent superinfection. Also of note the phage encodes a member of the hydrolase 2 superfamily implicated in bacterial cell wall hydrolysis. The nearest database phage sequence was PHAGE_Escher_phAPEC8 that infects avian pathogenic E. coli and is also a member of the Myoviridae (Tsonos et al., ).

Bacteriophage Host Range and Efficiency of Plating

The host range of five different phages isolated from freshwater, including phage ZCKP1 were tested on bacteria that were isolated from diabetic patients suffering from osteomyelitis. The ZCKP1 phage was capable of producing lysis zones (&#x;20 plaques) on 15 out of 21 K. pneumoniae isolates, 5 out of 18 P. mirabilis isolates and 9 out of 30 E. coli isolates, while other phages did not display a comparable spectrum of activity against the K. pneumoniae isolates (Table 1). A range of EOP for ZCKP1 phage was observed against different species of Enterobacteriacae (Supplementary Table 2). For K. pneumoniae seven phages demonstrated EOPs similar to the multidrug resistant host strain. For P. mirabilis, all susceptible strains showed EOP , whereas for E. coli six strains supported replication with EOPs approaching that of the permissive K. pneumoniae hosts (Table 2).

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Table 1. Lytic activity of isolated phages against K. pneumoniae and other selected members of the Enterobacteriaceae.

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Table 2. Efficiency of plating of phage ZCKP1 against different species of Enterobacteriacae.

Frequency of BIMS

BIMs were recovered following high multiplicity infections () of host bacteria K. pneumoniae, P. mirabilis and E. coli with bacteriophage ZCKP1 at 37°C. Mutational frequencies of × 10&#x;5 ± × 10&#x;4 and × 10&#x;5 ± × 10&#x;5 were determined for Klebsiella and E. coli, respectively where K. pneumoniae KP1 alone exhibited a lower frequency of 5 × 10&#x;6 ± × 10&#x;6.

In vitro Characterization of Phage ZCKP1

A single-step growth curved demonstrated bacteriophage virions were naturally released from bacterial cells after 30 min: the latent period which is the time taken for phages to be assembled and released after infection. However, viruses were assembled 10 min before. This was indicated by eclipse period that was estimated to be 20 min, as chloroform aids new phage particles to free from bacterial cell wall (Figure 2). Burst size was estimated to be  virions per single bacterium.

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Figure 2. Single step growth curve. Gray dashed line represents nascent phage without chloroform addition (PFU/ml), while the black line represents phages released after chloroform addition (PFU/ml).

The infection and lysis characteristics of phage ZCKP1 were estimated at different MOIs, over a period of 3 h (Figures 3A&#x;C) in a growing culture of K. pneumoniae KP/01 (Figures 3A&#x;C). K. pneumoniae KP/01 was lysed by phage ZCKP1 at each MOI tested but the MOI of reduced the viable bacteria from log10 CFU/ml to below the limit of detection at 37°C by 2 h (Figures 3A&#x;C). Under these circumstances the reductions in bacterial count were not accompanied by a measurable rise in phage titer (Figure 3C). Phage replication was observed at lower MOI, which coincided with the commencement of the fall in viable count.

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Figure 3. In vitro activity of phage ZCKP1 at 37°C. Panels show bacterial counts and phage titers of K. pneumoniae KP/01 infected with ZCKP1 at: (A) MOI ; (B) MOI 1; (C) MOI Black solid line represents viable count of K. pneumoniae KP/01 infected with phage (CFU/ml); Gray solid line represents K. pneumoniae KP/01 uninfected control (CFU/ml); black dashed line represents phage infective centers (PFU/ml) and gray dashed line represents nascent phage (PFU/ml).

Bacteriophage Activity Against K. pneumoniae Established in Biofilms

A single application of ZCKP1 to established biofilms of K. pneumoniae KP/01 resulted in a reduction crystal violet stainable biofilm content (P ; Figure 4A) and the percentage of viable cells observed by MTT staining (P ; Figure 4C) after 4 h. The most effective treat represented the highest MOI (50 PFU/CFU). However, following this disruption there was recovery in biofilm estimates accompanied by a recovery in cell viability. Multiple treatments of phage ZCKP1 on established K. pneumoniae KP/01 biofilms at 4 h intervals resulted in significant reductions in biofilm content and prevented the recovery of cell viability throughout the 24 h period of the experiment (P ; Figures 4B,D).

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Figure 4. Phage treatments of K. pneumoniae KP/01 biofilms. Panels (A) and (B) show the effect of phage treatment on preformed biofilms determined by crystal violet staining and solubilization estimates of biomass: (A) single treatment with phage ZCKP1; (B) with multiple treatments with phage ZCKP1 using different MOIs. White columns represent untreated control; light gray columns represent a starting MOI of 5; dark gray columns represent a starting MOI of 10 and solid black columns represent a starting MOI of Panels (C) and (D) show bacterial counts in biofilms determined using an MTT assay, (C) single treatment with phage ZCKP1 bacteriophage or (D) with multiple treatments with phage ZCKP1 bacteriophage using different MOIs: Light gray columns represent a starting MOI of 50; dark gray columns represent a starting MOI of 10 and solid black columns represent a starting MOI of 5. *P (brackets specify comparisons between groups).

Bacteriophage Temperature and pH Stability

The stability of phage ZCKP1 at different temperatures and pH values was investigated (Figures 5A,B). Phage titers were stable, at approximately 109 PFU/ml, for 1 h at temperatures of 45 and 55°C. The phage titer decreased after 40 min at 65°C to 108 PFU/ml, and continued to decline below 107 PFU/ml after 1 h. A significant decline (P ) was observed when phages were incubated at 75 and 85°C. However, phage could still be recovered after 1 h at 75°C at a titer of 103 PFU/ml. Phage could not be recovered after 40 min at 85°C. Acidic pH of 6 significantly (P ) reduced the phage stability after 1 h. The optimum stability was observed to be pH 6 but persisted at alkaline pH values to pH 9 (Figure 5B).

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Figure 5. The stability of phage ZCKP1 at (A) different temperatures (45°C: circle, 55°C: square, 65°C: triangle, 75°C: inverted triangle and 85°C: diamond) and (B) pH values. Results are shown as means ± Standard error.

Discussion

K. pneumoniae is an enteric pathogen that causes pneumonia and wound infections (Podschun and Ullmann, ). The effectiveness of antibiotics to treat such infections has been reduced significantly in recent years due to the increasing numbers of antibiotic-resistant bacteria, and as a result morbidity and mortality remain high. Antibiotic resistance is a growing public health threat for which the use of bacteriophage as an alternative to antibiotics may be considered to combat MDR infections. In particular phage therapy has also been considered a promising approach to eliminate diabetic foot ulcer after infection by MRSA in human subjects (Fish et al., ). In order to get the maximum benefits of bacteriophage based therapies, it is important to determine the characteristics of individual bacteriophages so that treatments can be tailored for the situation where treatment is to be applied. Moreover, it is crucial to ensure that phages selected do not have the capacity to transfer resistance or pathogenic traits to the resident microbiota (Abedon and Thomas-Abedon, ).

Antibiotics that were previously effective in the elimination of diabetic foot infections are now less effective. The K. pneumoniae KP/01 isolate recorded here shows resistance to levofloxacin, fluoroquinolone and was identified as a ceftazidime-resistant K. pneumoniae (CSKP). Ceftazidime is a cephalosporin antibiotic that can be degraded by extended spectrum beta lactamases (ESBL) that include SHV, TEM, CTX and YOU types (Sougakoff et al., ; Urban et al., ). K. pneumoniae KP/01 also showed resistance to the cephalosporin cefepime. As a clinical multiple drug resistant bacteria the KP/01 isolate was an ideal host for this study (Sougakoff et al., ).

Both morphological analysis and genome size confirmed that bacteriophage ZCKP1 belonged to the Caudovirales order with typical features of Myoviridae. It had an icosahedral head, a contractile tail with base plates showing tail fibers and spikes in addition to a collar. The genome of kb, differs in size to the 45 kbp of KLPN1 phage previously reported as isolated against K. pneumoniae (Hoyles et al., ). Bacteriophage ZCKP1 demonstrated a broad lytic profile covering a variety of bacterial pathogens including K. pneumoniae, Proteus and E. coli that all contribute to osteomyelitis cases and were isolated from patients with diabetic foot.

In vitro studies of potential therapeutic bacteriophages ensures only the most effective phages progress to clinical trials based on their capability to lyse pathogens in planktonic and biofilm formations with wide host range coverage. Phage ZCKP1 was shown to be highly effective at reducing K. pneumoniae counts in vitro and proved to be stable at high temperatures and over a wide pH range. Phage ZCKP1 was also effective against other members of Enterobacteriacae that cause osteomyelitis, which contributes to the therapeutic potential. With the application of high concentrations of bacteriophages (MOI of ), ZCKP1 was demonstrated to reduce K. pneumoniae without producing new phages. This is an established phenomenon called lysis from without, where many phages become absorbed to bacterial cells causing lysis without release of new phage (Abedon, ). In addition, a single high dose applied in a clinical situation may enable the human immune system to overcome reduced numbers of pathogens by working synergistically with the phage. Even with lower doses of phage, the rate of development of resistance to bacteriophages is approximately fold lower than the rate of the development of antibiotic resistance (Carlton, ). The conditions of application and the influence of immune system can vary so the action of a particular phage must be considered before therapeutic use (O&#x;Flynn et al., ; Lu and Koeris, ). In this context the mutation frequencies determined at high MOI applications would dictate the use of phage cocktails, and possibly the availability of reserve phage. Developing a cocktail of isolated lytic phages may increase the efficacy of bacteriophages to lyse multiple hosts and reduce the frequency that resistant strains may emerge.

Klebsiella are able to form thick biofilms on tissues and on medical implants making them more resistant than free-living planktonic cells to antibacterial agents and have reduced susceptibility to antibiotics (Calhoun and Manring, ). Phage ZCKP1 treatment of K. pneumoniae KP/01 biofilms was shown to be an effective method for biofilm reduction, although repeated treatments were required to prevent regrowth. Reductions in biofilm biomass have been attributed to the action of a soluble exopolysaccharide depolymerase (Cornelissen et al., ). These enzymes have the ability to disrupt the capsule of Klebsiella making it more susceptible to antibacterial agents (Hughes et al., ; Kesik-Szeloch et al., ). The nucleotide sequence of phage ZCKP1 revealed enzyme activities consistent with polysaccharide modification, However, the presence of wcaM could influence exopolysaccharide structure to adversely affect biofilm integrity when embedded bacteria become phage infected.

Previously reported phage treatments of K. pneumoniae biofilms include: a phage belonging to the Podoviridae family (Chhibber et al., ); a Siphoviridae named bacteriophage Z (Jamal et al., ) and Myoviridae phages (Kesik-Szeloch et al., ). Of these, the Myoviridae are likely the most promising as they represent virulent bacteriophage that do not mobilize and transfer genetic information. The gene sequence of phage ZCKP1 suggests that it does indeed fall into this category. Four genes associated with tellurite resistance were observed but are not used for antimicrobial therapy. Tellurite resistance is often associated with colicin and phage resistance phenotypes (Taylor and Summers, ), and likely extends this advantage to the virus infected cell as insurance against superinfection.

Conclusion

Phage ZCKP1 has been fully characterized in vitro and shows excellent potential to be used as a therapeutic agent against K. pneumoniae infections of diabetic foot. It can reduce the bacterial pathogen in both planktonic and biofilms and is extremely stable over a range of pH and temperatures. Therapeutic trials are needed to confirm its potential in vivo.

Data Availability

All data generated or analyzed during this study are included in this published article and are available from the corresponding author. Nucleotide sequences appear in the NCBI public database under the GenBank accession number MH

Author Contributions

AE-S: primary responsibility for design of the work. OT and AE-S: substantial contributions to the design of the work and analysis and interpretation of the data. OT, PC, IC, and AE-S: drafting the work and revising it critically for important intellectual content. OT, PC, IC, and AE-S: final approval of the version to be published.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This research was supported by Zewail City of Science and Technology. This work was also supported by the Biotechnology and Biological Sciences Research Council (grant number BB/GCRF-IAA/15).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles//fmicb/full#supplementary-material

Abbreviations

BIMs, bacteriophage insensitive mutants; IC, phage infective centers; MOI, multiplicity of infection; MTT, 3-(4, 5-dimethylthiazolyl)-2, 5-diphenyltetrazolium bromide.

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Keywords:Klebsiella, bacteriophage, ulcer, diabetes, biofilm, osteomyelitis

Citation: Taha OA, Connerton PL, Connerton IF and El-Shibiny A () Bacteriophage ZCKP1: A Potential Treatment for Klebsiella pneumoniae Isolated From Diabetic Foot Patients. Front. Microbiol. doi: /fmicb

Received: 03 October ; Accepted: 20 August ;
Published: 11 September

Copyright © Taha, Connerton, Connerton and El-Shibiny. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Ayman El-Shibiny, [email protected]

Sours: https://www.frontiersin.org/articles//fmicb/full
KLEBSIELLA PNEUMONIAE

What Is a Klebsiella Pneumoniae Infection? Symptoms, Causes, Diagnosis, Treatment, and Prevention

Complications of a Klebsiella Pneumoniae Infection

If K. pneumoniae gets into other areas of the body, it can cause a range of different illnesses.

These include:

  • Pneumonia When K. pneumoniae enters the respiratory tract, it can lead to bacterial pneumonia, or an infection of the lungs. Symptoms include chest pain when you breathe or cough, fever and chills, shortness of breath, fatigue, a cough that may produce phlegm, and changes in mental awareness. It is most serious in older adults, young children, and people with a compromised immune system.
  • Bloodstream infections Pneumoniae that enters the bloodstream can cause bacteremia, or an infection of the blood. Bacteremia needs to be treated right away, as these infections can progress to sepsis and septic shock, which can turn deadly. If you’ve recently had a medical or dental procedure or are in the hospital and experience a sudden fever and chills, tell your doctor right away.
  • Urinary tract infections (UTI) When pneumonia enters the urinary tract, it can lead to a UTI. A UTI can affect any part of the urinary system, including the urethra, kidneys, bladder, and ureters. Symptoms include a strong, frequent need to urinate, burning sensation during urination, pelvic pain, and cloudy, bloody, or strong-smelling urine. Women are at a greater risk of getting a UTI than men.
  • Wound and surgical site infections If K. pneumoniae enters a break in the skin, it can lead to a skin or soft tissue infection. Typically, this happens with wounds caused by injury or after surgery. Symptoms can include fever, blisters, fatigue, and pain at the wound or surgical site.
  • Meningitis Bacterial meningitis can occur when pneumoniae enters the membranes surrounding the brain and spinal cord. It is a very serious infection that can be life-threatening. The bacteria can cause the tissues around the brain to swell, interfering with blood flow. This can result in paralysis or stroke. Symptoms, including high fever, headaches, and stiff neck, come on quickly, usually within 24 hours of infection. If left untreated, bacterial meningitis can lead to death.
Sours: https://www.everydayhealth.com/klebsiella-pneumoniae/guide/

For pneumoniae treatment klebsiella

Klebsiella pneumoniae pneumonia

Klebsiella pneumoniae is an uncommon cause of community-acquired pneumonia except in alcoholics. Klebsiella may mimic pulmonary reactivation tuberculosis because it presents with hemoptysis and cavitating lesions. Klebsiella pneumoniae is a difficult infection to treat because of the organism's thick capsule. Klebsiella is best treated with third- and fourth-generation cephalosporins, quinolones, or carbapenems. Monotherapy is just as effective as a combination treatment in Klebsiella pneumoniae because newer agents are used. In the past, older agents with less anti-Klebsiella activity were needed for effective treatment. The patient we present was initially thought to have pulmonary tuberculosis, and when found to have Klebsiella pneumoniae, the suggested treatment was monotherapy with ceftriaxone. The patient was treated parenterally initially, and then was treated for 3 weeks with oral ofloxacin.

Sours: https://pubmed.ncbi.nlm.nih.gov//
Klebsiella pneumoniae - an Osmosis Preview

What to know about Klebsiella pneumoniae

Klebsiella pneumoniae is a type of bacteria that can cause a range of infections. These usually develop in hospital settings.

People have K. pneumoniae in their digestive tracts. When the bacteria spread to other parts of the body, they can cause a variety of infections, including:

  • urinary tract infections
  • skin and wound infections
  • liver abscesses
  • pneumonia
  • blood infections
  • meningitis

Keep reading to learn more about the causes, symptoms, and treatments of K. pneumoniae infections.

Causes

There are many types of K. pneumoniae bacteria. Some have capsules surrounding their cells, and others do not.

Researchers have currently identified . Klebsiella bacteria without capsules are less infectious than those with capsules.

Humans are the carriers of K. pneumoniae in the environment, but most people will not develop an infection. People with weakened immune systems, due to medications or medical conditions, have a higher risk.

Researchers that some populations carry more of this type of bacteria, including people of Chinese ethnicity and people with alcohol use disorder.

In some populations, the disease is more likely to cause certain infections. K. pneumoniae are the cause of hospital-acquired pneumonia in the United States, for example.

Meanwhile, in Western regions, Klebsiella rarely causes meningitis. However, in Taiwan, K. pneumoniae infection is a leading cause, responsible for about of bacterial meningitis cases in adults.

Some people develop K. pneumoniae meningitis from liver abscesses. The bacteria from the abscess can travel from the liver to the central nervous system.

In addition, catheters and tools in medical procedures can transmit K. pneumoniae into the urinary tract, the bloodstream, and wounds.

Symptoms

Different types of infection with K. pneumoniae can cause different symptoms, which may resemble those of other bacterial infections.

If a doctor notices that a bacterial infection persists after the initial treatment, they may order tests to identify the specific bacteria responsible. The results help them choose the most appropriate antibiotic treatment.

Anyone who suspects that they have a urinary tract infection, pneumonia, meningitis, or cellulitis — all of which can result from K. pneumoniae — should consult a doctor right away.

The table below lists common symptoms of these conditions.

Treatment

Doctors treat K. pneumoniae infections with antibiotics. When an infection is hospital-associated, doctors use a class of antibiotics called carbapenems until results of sensitivity testing are available.

If a doctor suspects that the bacteria have developed antibiotic resistance, they can order tests to determine how sensitive the bacteria are to specific antibiotics, before selecting the most effective option.

It may be challenging for doctors to treat K. pneumoniae infections because increasingly fewer antibiotics are effective. Most recently, for example, some K. pneumoniae have to carbapenems.

A doctor may prescribe a combination of antibiotics. One observed lower mortality rates in people with bacteremia from K. pneumoniae who had received a combination of the antibiotics colistin, meropenem, and tigecycline.

When a person develops pneumonia from K. pneumoniae, doctors a 2-week treatment with third- or fourth-generation cephalosporin, a fluoroquinolone, or one of these antibiotics in combination with an aminoglycoside.

People who are allergic to penicillin require a course of aztreonam or a quinolone.

Diagnosis

Doctors usually diagnoseKlebsiella infections by examining either a sample of infected tissue or a sample of:

Sometimes doctors order medical imaging tests, including:

  • ultrasounds
  • X-rays
  • CT scans

Once the doctor confirms the diagnosis, they may run sensitivity tests to determine which antibiotic will most effectively treat the infection.

When to see a doctor

Anyone who suspects that they have a K. pneumoniae infection should seek medical attention right away.

If any infection persists after home care or an initial course of antibiotics, it is important to seek medical attention. The doctor may ask for additional testing to check the susceptibility of the bacteria to antibiotics.

Is it contagious?

K. pneumoniae infection is . A person must come into contact with the bacteria, which do not spread through the air.

In hospitals, K. pneumoniae can spread through person-to-person contact. People may also come into contact with the bacteria through environmental exposure, though this occurs less often.

An individual may come into contact with this type of bacteria through:

  • ventilators, or breathing machines
  • intravenous catheters
  • urinary catheters
  • open wounds

Healthy family members of people with K. pneumoniae infections have a low risk of acquiring the infection.

However, taking every hygiene precaution is essential. Hand hygiene remains the best defense against K. pneumoniae infection.

Prognosis

When doctors identify K. pneumoniae in samples quickly and prescribe the appropriate antibiotics right away, the prognosis improves. However, delays in diagnosis and testing are common, and this can lead to a less favorable .

The prognosis for people with pneumonia from K. pneumoniae is often poor. Even when doctors choose the appropriate antibiotic therapy, mortality rates are .

People with other diseases, such as diabetes, older adults, and people with compromised immune systems have the highest risks of mortality.

In people with pneumonia from these bacteria, the infection may impede lung function in the long term, possibly for months.

Summary

K. pneumoniae infections typically develop in hospital settings. People with weakened immune systems and chronic conditions have the highest risk.

K. pneumoniae have developed resistance to many antibiotics, and doctors may find it challenging to treat K. pneumoniae infections. However, testing the sensitivity of the bacteria in blood or tissue samples can help them identify the most effective course of treatment.

People with K. pneumoniae infections may transmit the bacteria to others. Taking every hygiene precaution, especially hand washing, is the best way to prevent K. pneumoniae infections from spreading.

Sours: https://www.medicalnewstoday.com/articles/klebsiella-pneumoniae

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