Since the hypermucoviscous variants of Klebsiella pneumoniae were first reported, many cases of primary liver abscesses and other invasive infections caused by this pathogen have been described worldwide. Hypermucoviscosity is a phenotypic feature characterized by the formation of a viscous filament ≥5 mm when a bacterial colony is stretched by a bacteriological loop; this is the so-called positive string test. Hypermucoviscosity appears to be associated with this unusual and aggressive type of infection, and therefore, the causal strains are considered hypervirulent. Since these first reports, the terms hypermucoviscosity and hypervirulence have often been used synonymously. However, new evidence has suggested that hypermucoviscosity and hypervirulence are 2 different phenotypes that should not be used synonymously. Moreover, it is important to establish that a negative string test is insufficient in determining whether a strain is or is not hypervirulent. On the other hand, hypervirulence- and hypermucoviscosity-associated genes must be identified, considering that these phenotypes correspond to 2 different phenomena, regardless of whether they can act in synergy under certain circumstances. Therefore, it is essential to quickly identify the genetic determinants behind the hypervirulent phenotype to develop effective methodologies that can diagnose in a prompt and effective way these hypervirulent variants of K. pneumoniae.

The Klebsiella pneumoniae complex comprises seven K. pneumoniae- related species, including K. variicola . K. variicola is a versatile bacterium capable of colonizing different hosts such as plants, humans, insects and animals. Currently, K. variicola is gaining recognition as a cause of several human infections; nevertheless, its virulence profile is not fully characterized. The clinical significance of K. variicola infection is hidden by imprecise detection methods that underestimate its real prevalence; however, several methods have been developed to correctly identify this species. Recent studies of carbapenemase-producing and colistin-resistant strains demonstrate a potential reservoir of multidrug-resistant genes. This finding presents an imminent scenario for spreading antimicrobial resistant genes among close relatives and, more concerningly, in clinical and environmental settings. Since K. variicola was identified as a novel bacterial species, different research groups have contributed findings elucidating this pathogen; however, important details about its epidemiology, pathogenesis and ecology are still missing. This review highlights the most significant aspects of K. variicola , discussing its different phenotypes, mechanisms of resistance, and virulence traits, as well as the types of infections associated with this pathogen.

ObjectivesTo characterize the microbiological, molecular and epidemiological data of an outbreak of carbapenem-resistant Enterobacteriaceae (CRE) in a tertiary-care hospital in Mexico.MethodsFrom September 2014 to July 2015, all CRE clinical isolates recovered during an outbreak in the Hospital Civil "Fray Antonio Alcalde" in Jalisco, Mexico were screened for antimicrobial susceptibility, carbapenemase production, carbapenemase-encoding genes, and plasmid profiles. Horizontal transfer of imipenem resistance; and clonal diversity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST); as well as biofilm production and the presence of 14 virulence genes were analyzed in selected isolates.ResultsFifty-two carbapenem-resistant isolates corresponding to 5 species were detected, i.e., Klebsiella pneumoniae (n = 46), Enterobacter cloacae (n = 3), Escherichia coli (n = 1), Providencia rettgeri (n = 1) and Citrobacter freundii (n = 1) with carbapenemase encoding genes blaNDM-1 (n = 48), blaVIM (n = 3), blaIMP (n = 1) and blaKPC (n = 1) detected in these isolates.The blaNDM-1 gene was detected in plasmids from 130- to 170-kb in K. pneumoniae (n = 46); E. cloacae (n = 3), E. coli (n = 1) and P. rettgeri (n = 1). The transfer of plasmids harboring the blaNDM-1 gene was obtained in eight transconjugants. One plasmid restriction pattern was detected, with the blaNDM-1 identified in different restriction fragments.Predominant clone A of K. pneumoniae isolates archived 28/46 (60%) isolates and belongs to ST392. Besides, ST307, ST309, ST846, ST2399, and ST2400 were detected for K. pneumoniae; as well as E. cloacae ST182 and E. coli ST10.The fimA and uge genes were more likely to be identified in K. pneumoniae carbapenem-susceptible isolates (p = <0.001) and biofilm production was more liable to be observed in carbapenem-resistant isolates (p = <0.05).ConclusionsFour Enterobacteriaceae species harboring the blaNDM-1 gene were detected in a nosocomial outbreak in Mexico; horizontal transfer and strain transmission were demonstrated for the blaNDM-1 gene. Given the variation in the size of the plasmid harboring blaNDM-1, complex rearrangements must also be occurring.

BackgroundKlebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.ResultThis work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.ConclusionsThis multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0396-6) contains supplementary material, which is available to authorized users.

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two ␤-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single ␤-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 ␤-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5␣. Sequencing of the bla TLA-1 gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A ␤-lactamases:70 SXXK, 130 SDN, and 234 KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A ␤-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A ␤-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 ␤-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum ␤-lactamase of Ambler class A.The main mechanism of resistance to ␤-lactam antibiotics in members of the family Enterobacteriaceae is the production of ␤-lactamases (21, 35). Expanded-spectrum cephalosporins (cefotaxime, ceftazidime) have been specifically designed to resist degradation by the older broad-spectrum ␤-lactamases such as TEM-1, TEM-2, and SHV-1. With the use of these antibiotics in vivo, extended-spectrum ␤-lactamases (ESBLs) have been selected; these ESBLs most often are mutants of these older enzymes and carry a limited number of amino acid substitutions (G. Jacoby and K. Bush, http://www.lahey.org/studies /webt.htm). There is also a small but growing family of plasmid-mediated ESBLs that are not related to TEM or SHV ␤-lactamases, such as 10,14,15) and Toho (17,23), that preferentially hydrolyze cefotaxime and that belong to Ambler class A. In addition, there has been a worldwide emergence of novel ␤-lactamases, mainly among members of the family Enterobacteriaceae, that hydrolyze expanded-spectrum ␤-lactams. While they maintain the main properties of the class A ␤-lactamases, they are not closely related to the TEM, SHV, or CTX-M families of ␤-lactamases. Most of these ESBLs are plasmid mediated and include the PER-1, PER-2, VEB-1, CblA, and CepA enzymes. These ␤-lactamases are not species specific, since they have also been isolated from clinically significant gram-negative species that are not mem...

Thirty-one strains of Klebsiella pneumoniae (including 10 duplicates) from 21 septicemic pediatric patients (age, <2 months) were studied during a 4-month period (June to October 1996) in which the fatality rate was 62% (13 of 21). These isolates identified by the API 20E system yielded the same biotype. Pulsed-field gel electrophoresis experiments revealed the same clone in 31 strains. The isolates were multidrug-resistant but were still susceptible to ciprofloxacin, imipenem, and cefoxitin. A 135-kb plasmid was harbored in all of the isolates. No transconjugants were obtained that were resistant to ampicillin, cefotaxime, tetracycline, or gentamicin. Isoelectric focusing for ␤-lactamases was performed on all strains, and three bands with pIs of 5.4, 7.6, and 8.2 were obtained. Of these, the pI 8.2 ␤-lactamase had an extended-spectrum ␤-lactamase phenotype. PCR amplification of both TEM-and SHV-type genes was obtained. The sequence analysis of the SHV PCR product indicated a mutation corresponding to the SHV-5 ␤-lactamase.Klebsiella pneumoniae is an important hospital-acquired pathogen with the potential of causing severe morbidity and mortality in pediatric patients. Several outbreaks of infection caused by K. pneumoniae isolates that are simultaneously resistant to broad-spectrum cephalosporins and aminoglycosides have been widely reported (8,9,18,21). Some of these multidrug-resistant isolates produce extended-spectrum ␤-lactamases (ESBLs) that are able to hydrolyze expanded-spectrum cephalosporins (e.g., ceftriaxone, cefotaxime, and ceftazidime), aztreonam, and related oxyimino-␤-lactams. Most of these enzymes are TEM-or SHV-type ␤-lactamases in which the substitution of one or more amino acids has altered the configuration of the active site (7,14,19). Most of the plasmids determining ESBLs are large (Ն80 kb) and encode multiple resistances (10). Little is known about the ESBL-producing Enterobacteriaceae in Mexico (23). The present work is a characterization of 31 ESBL-producing K. pneumoniae isolates from a nosocomial outbreak occurring in a hospital in Cuernavaca, Mexico. MATERIALS AND METHODSPatients and bacterial strains. The General Hospital in the state of Morelos is a secondary-care facility with 100 beds. The neonatal intensive care unit has 8 beds. An outbreak was suspected in the neonatal intensive care unit due to an increased number of isolates of K. pneumoniae from blood cultures during a 4-month period. A retrospective epidemiological investigation included 74 children less than 2 months old hospitalized from June to October 1996.Twenty-one clinical isolates were from blood; another 10 duplicate strains were from sites other than blood (urine and catheter tip) corresponding to 10 patients. In total there were 31 clinical isolates identified as K. pneumoniae by the API 20E system (BioMerieux, Merck).Susceptibility testing. The antimicrobial susceptibility was initially determined with the MicroScan (Dade) system, using the Combo 14 panel. Subsequently, MICs of several ␤-lactams were determine...

During 2003, 40 carbapenem-resistant Pseudomonas aeruginosa clinical isolates collected in a Mexican tertiary-care hospital were screened for metallo-␤-lactamase production. Thirteen isolates produced IMP-15, and 12 had a single pulsed-field gel electrophoresis pattern. The bla IMP-15 gene cassette was inserted in a plasmid-borne integron with a unique array of gene cassettes and was named In95.

Klebsiella variicola is considered an emerging pathogen in humans and has been described in different environments. K. variicola belongs to Klebsiella pneumoniae complex, which has expanded the taxonomic classification and hindered epidemiological and evolutionary studies. The present work describes the molecular epidemiology of K. variicola based on MultiLocus Sequence Typing (MLST) developed for this purpose. In total, 226 genomes obtained from public data bases and 28 isolates were evaluated, which were mainly obtained from humans, followed by plants, various animals, the environment and insects. A total 166 distinct sequence types (STs) were identified, with 39 STs comprising at least two isolates. The molecular epidemiology of K. variicola showed a global distribution for some STs was observed, and in some cases, isolates obtained from different sources belong to the same ST. Several examples of isolates corresponding to kingdom-crossing bacteria from plants to humans were identified, establishing this as a possible route of transmission. goeBURST analysis identified Clonal Complex 1 (CC1) as the clone with the greatest distribution. Whole-genome sequencing of K. variicola isolates revealed extended-spectrum β-lactamase- and carbapenemase-producing strains with an increase in pathogenicity. MLST of K. variicola is a strong molecular epidemiological tool that allows following the evolution of this bacterial species obtained from different environments.