Colistin Resistance Caused by Inactivation of the MgrB Regulator Is Not Associated with Decreased Virulence of Sequence Type 258 KPC Carbapenemase-Producing Klebsiella pneumoniae

Using a Galleria mellonella animal model, we compared the virulence of two sequence type 258 (ST258) KPC-producing Klebsiella pneumoniae strains, which were representative of the two clades of this clonal lineage, with that of isogenic colistin-resistant mgrB mutants. With both strains, the mgrB mutants did not exhibit modification in virulence. In the G. mellonella model, the clade 1 strain (capsular type cps-1 [wzi29, producing KPC-2]) was significantly more virulent than the clade 2 strain (capsular type cps-2 [wzi154, producing KPC-3]).     

Genetic Factors Associated with Elevated Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae

In the United States, the most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is the production of a Klebsiella pneumoniae carbapenemase (KPC). KPC-producing isolates often exhibit a range of carbapenem MICs. To better understand the factors that contribute to overall carbapenem resistance, we analyzed 27 KPC-producing K. pneumoniae isolates with different levels of carbapenem resistance, 11 with low-level (i.e., meropenem or imipenem MIC ≤ 4 μg/ml), 2 with intermediate-level (i.e., meropenem and imipenem MIC = 8 μg/ml), and 14 with high-level (i.e., imipenem or meropenem MIC ≥ 16 μg/ml) carbapenem resistance, that were received from throughout the United States. Among 14 isolates that exhibited high-level carbapenem resistance, Western blot analysis indicated that 10 produced an elevated amount of KPC. These isolates either contained an increased bla_KPC gene copy number (n = 3) or had deletions directly upstream of the bla_KPC gene (n = 7). Four additional isolates lacked elevated KPC production but had high-level carbapenem resistance. Porin sequencing analysis identified 22 isolates potentially lacking a functional OmpK35 and three isolates potentially lacking a functional OmpK36. The highest carbapenem MICs were found in two isolates that lacked both functioning porins and produced elevated amounts of KPC. The 11 isolates with low-level carbapenem resistance contained neither an upstream deletion nor increased bla_KPC copy number. These results suggest that both bla_KPC copy number and deletions in the upstream genetic environment affect the level of KPC production and may contribute to high-level carbapenem resistance in KPC-producing K. pneumoniae, particularly when coupled with OmpK36 porin loss.The occurrence of Gram-negative bacterial infections that are resistant to extended-spectrum β-lactam antimicrobial agents forces clinicians to rely on carbapenems as a “last resort” to combat these resistant pathogens. However, as carbapenems are more frequently utilized, an increasing number of bacteria with various mechanisms of resistance to this class of antimicrobial agents are identified. The most widespread resistance mechanisms include the production of a carbapenemase and the combination of porin loss with the production of either an AmpC enzyme or an extended-spectrum β-lactamase (4, 15). Klebsiella pneumoniae carbapenemase (KPC), an Ambler class A β-lactamase that can hydrolyze most β-lactam agents, including carbapenems, is now the most prevalent carbapenemase found among clinical Gram-negative isolates in the United States (22).KPC was first reported in a K. pneumoniae isolate from North Carolina in 1996 (28). However, recent reports indicate that KPC-producing Gram-negative isolates are being identified throughout the United States as well as parts of Europe, Asia, and South America (13, 20, 22). Although these β-lactamases occur most commonly in K. pneumoniae, they have also been identified in other members of the Enterobacteriaceae family and in Pseudomonas and Acinetobacter species (3, 21, 24, 26, 27). The bla_KPC gene is plasmid mediated and is carried on a Tn3-based transposon, Tn4401 (17), which may account for the high mobility of this resistance mechanism.KPC-producing isolates can exhibit a range of carbapenem MICs, thus making their detection a significant challenge for clinical laboratories. By using 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints and testing methods (1, 6), some KPC-producing isolates may be identified as susceptible to carbapenems. The clinical significance of carbapenem-susceptible isolates with elevated carbapenem MICs is unclear (6), and the cellular changes that may convert a susceptible KPC-producing isolate to one with MICs indicating resistance to carbapenem are not well described. From previous reports, we know that KPC production combined with porin loss can result in higher carbapenem MICs (10, 14, 29). This finding suggests that the KPC enzyme alone is not always sufficient to confer carbapenem resistance, as defined by the 2009 CLSI breakpoints.Other factors likely result in higher carbapenem MICs for KPC-producing isolates. For example, isolates with an increased expression of bla_KPC were previously shown to have increased rates of hydrolysis of imipenem and meropenem (14). Directly upstream of the bla_KPC gene is a nonconserved region of the Tn4401 transposon, located between the istB and the bla_KPC genes (17). Previous reports describe four isoforms in this variable region: Tn4401a contains a 100-bp deletion, Tn4401b contains no deletion (17), and isoforms with 215-bp (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ989640","term_id":"116263782","term_text":"DQ989640"}}DQ989640) and 255-bp (13) deletions were recently reported. Additional studies of this variable region suggest that the 100-bp deletion may result in a different −35 promoter region of the bla_KPC gene (11). Upstream deletions that affect the promoter may impact the level of bla_KPC expression and thus would influence the overall level of carbapenem resistance. Also, KPC-producing isolates may contain different levels of bla_KPC dosage based on the presence of multiple copies of a bla_KPC-carrying plasmid, multiple bla_KPC-carrying plasmids, or multiple copies of the bla_KPC gene located within the same plasmid (11). Increasing the bla_KPC gene copy number could result in increased enzyme production and higher carbapenem MICs. Understanding the impact of these factors may help to predict the potential for KPC-producing isolates susceptible to carbapenems to convert to isolates resistant to carbapenems.In this study, we examined genetic factors that may enhance the level of carbapenem resistance. We selected 27 KPC-producing K. pneumoniae isolates that were obtained from clinical patients in different areas of the country and exhibited a range of carbapenem MICs. These isolates were characterized by determining the sequences of the two main porins, OmpK35 and OmpK36 (9), examining levels of KPC production by Western blot analysis, comparing relative bla_KPC copy numbers using quantitative real-time PCR, and analyzing sequence variations in the genetic environment directly upstream of the bla_KPC gene.     

Community-Acquired Pyelonephritis in Pregnancy Caused by KPC-Producing Klebsiella pneumoniae

Carbapenem-resistant Enterobacteriaceae (CRE) usually infect patients with significant comorbidities and health care exposures. We present a case of a pregnant woman who developed community-acquired pyelonephritis caused by KPC-producing Klebsiella pneumoniae. Despite antibiotic treatment, she experienced spontaneous prolonged rupture of membranes, with eventual delivery of a healthy infant. This report demonstrates the challenge that CRE may pose to the effective treatment of common infections in obstetric patients, with potentially harmful consequences to maternal and neonatal health.     

Multiple Genetic Mutations Associated with Polymyxin Resistance in Acinetobacter baumannii

We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo. All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro.     

Transferrable Resistance to Tobramycin in Klebsiella pneumoniae and Enterobacter cloacae Associated with Enzymatic Acetylation of Tobramycin

Among gram-negative bacilli isolated from burn wound cultures, some strains of Enterobacteriaceae were resistant to tobramycin (minimal inhibitory concentration [MIC]≥ 20 μg/ml) but susceptible to gentamicin (MIC ≤ 5 μg/ml). One Klebsiella pneumoniae and two Enterobacter cloacae strains were selected for studies on their mechanisms of resistance to aminoglycoside antibiotics. Resistance to high concentrations of tobramycin (MICs of 25 to 50 μg/ml) was conjugally transferred to a susceptible Escherichia coli strain at rates of 1.2 × 10⁻⁴ to 2.8 to 10⁻⁴ per donor cell, suggesting that resistance is controlled by R factors. Resistances to tobramycin, kanamycin, and neomycin were cotransferred. Enzymatic activities were present that acetylated tobramycin, gentamicin, and kanamycin in osmotic lysates from the donor and transcipient strains. Enzymatic adenylylation of these aminoglycosides was not observed. The aminoglycoside-acetylating activities from K. pneumoniae and E. cloacae resembled kanamycin acetyltransferase (KAT) in their specificity for aminoglycoside substrates. Not all isolates of bacteria that produce KAT are resistant to tobramycin, but the factors that determine susceptibility or resistance to tobramycin in KAT-producing bacteria have not yet been established.     

Monomicrobial Necrotizing Fasciitis Caused by Aeromonas hydrophila and Klebsiella pneumoniae

Objective

To compare specific characteristics and clinical outcomes of monomicrobial necrotizing fasciitis caused by Aeromonas hydrophila and Klebsiella pneumoniae.

Material and Methods

Cases of monomicrobial necrotizing fasciitis caused by A. hydrophila (n = 11) and K. pneumoniae (n = 7) over an 8-year period were retrospectively reviewed. Differences in mortality, patient characteristics, clinical presentations, and laboratory data were compared between the A. hydrophila and the K. pneumoniae groups.

Results

The clinical signs and symptoms at the time of presentation did not differ significantly (p > 0.05) between the two groups. The A. hydrophila group had a significantly shorter interval between contact and admission (1.55 ± 0.52 vs. 5.14 ± 2.12 days, p < 0.001) and significant lower total white blood cell counts (10,245 ± 5,828 vs. 19,014 ± 11,370 cells/mm³, p < 0.045) than the K. pneumoniae group in the emergency room. Hepatic dysfunction was associated with mortality in patients with A. hydrophila infection, while diabetes mellitus was associated with mortality in patients with K. pneumoniae infection. Overall, 5 (45.5%) patients in the A. hydrophila group and 3 (42.8%) in the K. pneumoniae group died.

Conclusion

The initial clinical course of A. hydrophila monomicrobial necrotizing fasciitis was characterized by more rapidly progressive disease than that of the K. pneumoniae infection. Patients with hepatic dysfunction and necrotizing fasciitis should be suspected of having A. hydrophila infection, and diabetic patients with necrotizing fasciitis should be suspected of having K. pneumoniae infection initially.Key Words: Necrotizing fasciitis, Aeromonas hydrophila, Klebsiella pneumoniae     

Ertapenem-Containing Double-Carbapenem Therapy for Treatment of Infections Caused by Carbapenem-Resistant Klebsiella pneumoniae

We describe outcomes of patients with infections with carbapenem-resistant Klebsiella pneumoniae (CRKP) who received ertapenem-containing double-carbapenem therapy (ECDCT). Clinical success was observed in 7/18 (39%) patients overall: bloodstream infections, 3/7 (43%); pneumonia, 1/5 (20%); intraabdominal infections, 0/2 (0%); urinary tract infections, 2/3 (67%); and a skin and skin structure infection, 1/1 (100%). Microbiologic success was observed in 11/14 (79%) evaluable patients; 5/18 (28%) patients died. ECDCT may be effective for CRKP infections with limited treatment options.     

Acquisition of Broad-Spectrum Cephalosporin Resistance Leading to Colistin Resistance in Klebsiella pneumoniae

An extended-spectrum β-lactamase (ESBL)-producing and colistin-resistant Klebsiella pneumoniae clinical isolate was recovered from a patient who was treated with cefotaxime. This isolate harbored a bla_CTX-M-15 ESBL gene that was associated with an ISEcp1 insertion sequence. Transposition of that tandem occurred within the chromosomal mgrB gene, leading to inactivation of the mgrB gene and consequently to acquired resistance to colistin. We showed here a coselection of colistin resistance as a result of a broad-spectrum cephalosporin selective pressure.     

Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.     

Heteroresistance to Colistin in Klebsiella pneumoniae Associated with Alterations in the PhoPQ Regulatory System

A multidrug-resistant Klebsiella pneumoniae isolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activates pmrHFIJKLM expression responsible for l-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-type phoP gene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible subpopulation showed a partial deletion (25 bp) in the phoP gene compared to that in the colistin-resistant subpopulation. That deletion disrupted the reading frame of phoP, leading to a longer and inactive protein (255 versus 223 amino acids long). This is the first report showing the involvement of mutation(s) in PhoP in colistin resistance. Furthermore, this is the first study to decipher the mechanisms leading to colistin heteroresistance in K. pneumoniae.