Pietro E. Varaldo

Antibiotic Resistance Related to Biofilm Formation in Klebsiella pneumoniae

The Gram-negative opportunistic pathogen, Klebsiella pneumoniae, is responsible for causing a spectrum of community-acquired and nosocomial infections and typically infects patients with indwelling medical devices, especially urinary catheters, on which this microorganism is able to grow as a biofilm. The increasingly frequent acquisition of antibiotic resistance by K. pneumoniae strains has given rise to a global spread of this multidrug-resistant pathogen, mostly at the hospital level. This scenario is exacerbated when it is noted that intrinsic resistance to antimicrobial agents dramatically increases when K. pneumoniae strains grow as a biofilm. This review will summarize the findings about the antibiotic resistance related to biofilm formation in K. pneumoniae.

Streptococcus suis, an Emerging Drug-Resistant Animal and Human Pathogen

Streptococcus suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Recent studies and the analysis of sequenced genomes have been providing important insights into the S. suis resistome, and have resulted in the identification of resistance determinants for tetracyclines, macrolides, aminoglycosides, chloramphenicol, antifolate drugs, streptothricin, and cadmium salts. Resistance gene-carrying genetic elements described so far include integrative and conjugative elements, transposons, genomic islands, phages, and chimeric elements. Some of these elements are similar to those reported in major streptococcal pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae and share the same chromosomal insertion sites. The available information strongly suggests that S. suis is an important antibiotic resistance reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci. S. suis is thus a paradigmatic example of possible intersections between animal and human resistomes.

ICESp2905, the erm(TR)-tet(O) Element of Streptococcus pyogenes, Is Formed by Two Independent Integrative and Conjugative Elements

ABSTRACT In ICESp2905, a widespread erm(TR)- and tet(O)-carrying genetic element of Streptococcus pyogenes, the two resistance determinants are contained in separate fragments inserted into a scaffold of clostridial origin. ICESp2905 (∼65.6 kb) was transferable not only in its regular form but also in a defective form lacking the erm(TR) fragment (ICESp2906, ∼53.0 kb). The erm(TR) fragment was also an independent integrative and conjugative element (ICE) (ICESp2907, ∼12.6 kb). ICESp2905 thus results from one ICE (ICESp2907) being integrated into another (ICESp2906).

Two distinct genetic elements are responsible for erm(TR)-mediated erythromycin resistance in tetracycline-susceptible and tetracycline-resistant strains of Streptococcus pyogenes.

ABSTRACT In Streptococcus pyogenes, inducible erythromycin (ERY) resistance is due to posttranscriptional methylation of an adenine residue in 23S rRNA that can be encoded either by the erm(B) gene or by the more recently described erm(TR) gene. Two erm(TR)-carrying genetic elements, showing extensive DNA identities, have thus far been sequenced: ICE10750-RD.2 (∼49 kb) and Tn1806 (∼54 kb), from tetracycline (TET)-susceptible strains of S. pyogenes and Streptococcus pneumoniae, respectively. However, TET resistance, commonly mediated by the tet(O) gene, is widespread in erm(TR)-positive S. pyogenes. In this study, 23 S. pyogenes clinical strains with erm(TR)-mediated ERY resistance—3 TET susceptible and 20 TET resistant—were investigated. Two erm(TR)-carrying elements sharing only a short, high-identity erm(TR)-containing core sequence were comprehensively characterized: ICESp1108 (45,456 bp) from the TET-susceptible strain C1 and ICESp2905 (65,575 bp) from the TET-resistant strain iB21. While ICESp1108 exhibited extensive identities to ICE10750-RD.2 and Tn1806, ICESp2905 showed a previously unreported genetic organization resulting from the insertion of separate erm(TR)- and tet(O)-containing fragments in a scaffold of clostridial origin. Transferability by conjugation of the erm(TR) elements from the same strains used in this study had been demonstrated in earlier investigations. Unlike ICE10750-RD.2 and Tn1806, which are integrated into an hsdM chromosomal gene, both ICESp1108 and ICESp2905 shared the chromosomal integration site at the 3′ end of the conserved rum gene, which is an integration hot spot for several mobile streptococcal elements. By using PCR-mapping assays, erm(TR)-carrying elements closely resembling ICESp1108 and ICESp2905 were shown in the other TET-susceptible and TET-resistant test strains, respectively.

Lysozyme-Induced Inhibition of the Lymphocyte Response to Mitogenic Lectins'

Abstract Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 μg/ml for HL and 1 μg/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 μg/ml HL and 1 μg/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.

Unconventional Circularizable Bacterial Genetic Structures Carrying Antibiotic Resistance Determinants

Particular genetic structures which—though they lack their own recombinase genes— can be excised in circular form thanks to extensive direct repeats (DRs) flanking the DNA segment undergoing excision have recently been described in both Gram-negative and Gram-positive bacteria (1–6). They carry mostly antibiotic resistance genes. The earliest and the latest three of the above-noted studies were published in Antimicrobial Agents and Chemotherapy in 2006 (1) and 2012 (4–6). Although it is probably too early to consider such structures a new group of mobile elements, they are positively unlike conventional mobile genetic elements (MGEs) (plasmids, bacteriophages, integrative and conjugative elements [ICEs], or transposons) (7) and are here tentatively referred to as unconventional circularizable structures (UCSs). Reported UCSs and some putative UCSs are shown in Table 1. Besides excision, UCS integration in the repaired genetic context has been demonstrated experimentally, suggesting that, once excised, the DNA fragment can not only be lost but also undergo transposition (3). A recent study of eukaryotic genomes (Arabidopsis) hypothesized that intrachromosomal recombination of DRs having nontransposon sequences and subsequent insertion of the circular product may be the predominant mechanism of gene transposition (8). UCSs occurring in bacteria may play a similar role, which their frequent carriage of antibiotic resistance determinants makes even more intriguing. On the other hand, the resulting resistant phenotype could make those UCSs easier to find than UCSs devoid of resistance genes. The DRs acting in UCSs are usually long— up to more than 100 times longer than the well-established att sites acting in conventional MGEs (7)—and imperfect, and they may contain genes (of course, genes not involved in transposition). The encompassed DNA segments vary in length and often carry niche adaptation determinants. The recA gene has been shown to be dispensable for UCS excision/integration (2, 3, 6). A parasitic mobilization strategy via site-specific recombination and exploitation of the host trans-acting functions has been hypothesized (3), although the possibility of alternative homologous recombination pathways cannot be excluded, nor can the possibility that different UCSs have different mechanisms of excision/integration. An early report of a genetic structure apparently representing a UCS involved a circular minielement carrying the tetracycline resistance determinant tet(W) in the conjugative transposon TnB1230 of Butyrivibrio fibrisolvens (1). Remarkably, the minielement was detected in the transconjugants but not in the donor, suggesting that excision was dependent on host functions. Afterwards, two UCSs were characterized in enteric bacteria, one representing a defective prophage (2) and the other a microcin-encoding genomic island (3). Very recent studies of Gram-positive bacteria described UCSs consistently carrying antibiotic resistance genes. One, bearing the multidrug resistance gene cfr and containing the macrolide resistance gene erm(B) in the DRs, was reported in a methicillin-resistant Staphylococcus aureus isolate (4). Two more UCSs were described in streptococci, namely, Streptococcus suis and Streptococcus pneumoniae. The former (5), carried on an ICE, contains a number of antibiotic resistance genes: tet(O/W/ 32/O) and tet(40) (tetracycline), erm(B) (erythromycin), aadE (streptomycin), and aphA (kanamycin). The latter (6) is the wellknown MAS (macrolide-aminoglycoside-streptothricin) element, whose insertion distinguishes Tn1545/Tn6003 from Tn6002 (9, 10); again, the DRs contain the erm(B) gene. Of special interest is the involvement of erm(B) in recombination events concerning some UCSs. Besides the two erm(B)-containing DRs mentioned above (4, 6), erm(B)-containing DRs are likely to account for a deleted form (11) of Tn5398, the best-known erm(B)carrying element of Clostridium difficile (12). erm(B), one of the most prevalent and best-conserved antibiotic resistance genes in bacteria (http://faculty.washington.edu/marilynr/), may enable those UCSs that exploit it for integration to attain diverse, even phylogenetically distant, bacterial genomes. The antibiotic resistance determinants carried by UCSs are often freshly acquired genes for the host. This is true of tet(W) in B. fibrisolvens (13), of chromosomally located cfr in S. aureus (14), of tet(O/W/32/O) and tet(40) in S. suis (5, 15), and of aphA and sat4 (streptothricin) in the MAS element, when the clinical pneumococcus carrying it was originally isolated (16). It also applies to other instances where a UCS is suspected but was not expressly investigated. For example, when tet(W) was first described in Rothia, it was found in a region flanked by DRs containing a mef (macrolide efflux) gene (17). The cat (chloramphenicol acetyltransferase) gene was found in a spontaneously curable cargo DNA region flanked by DRs containing toxin/antitoxin genes when detected in Tn5253 of S. pneumoniae (18–20). In addition, erm(43), a new erm gene lately identified in Staphylococcus lentus, was found in an acquired DNA fragment flanked by DRs (21). The inherent instability of UCSs makes them unlikely to persist long as such in a given genetic context; rather, they will tend either to become stable (e.g., by sequence divergence between DRs or deletion of either DR) or to be lost (and possibly move to another genetic context). It is reasonable to assume that several resistance determinants have been acquired via UCSs and have later stabilized. The fact that UCSs are often carried by conventional MGEs might entail a mutual benefit, with UCSs contributing to prompt

Striking “Seesaw Effect” between Daptomycin Nonsusceptibility and β-Lactam Susceptibility in Staphylococcus haemolyticus

The expression “seesaw effect” was originally used by Sieradzki and Tomasz (7), and subsequently by others, to denote a frequently observed inverse relationship between evolving glycopeptide and β-lactam MICs in Staphylococcus aureus. The same expression has recently been revived in two interesting studies by Yang et al. (9) and Lee et al. (4) to signify a similar phenomenon, again seen in S. aureus, involving daptomycin instead of glycopeptides. On the other hand, it is well known that S. aureus strains progressively acquiring daptomycin nonsusceptibility during daptomycin exposure also exhibit progressively increasing vancomycin MICs (3, 6). Among coagulase-negative staphylococci, Staphylococcus haemolyticus, second only to Staphylococcus epidermidis in the frequency of its association with human infections (1), is unique in being predisposed to developing glycopeptide resistance and was the first Gram-positive pathogen to acquire such resistance in the 1980s (2). After exposure to increasing daptomycin concentrations, by a procedure successfully used with glycopeptides in previous studies in our laboratory (8), a stable clone with a daptomycin MIC of 4 μg/ml was obtained from a daptomycin-susceptible (MIC, 0.5 μg/ml) clinical isolate of S. haemolyticus. Vancomycin and teicoplanin MICs also increased from 4 and 8 μg/ml in the parent to 8 and 32 μg/ml in the laboratory derivative, respectively. The parent strain was both penicillin and methicillin resistant: penicillin and cefoxitin MICs were >256 μg/ml; molecular analysis disclosed a type I SCCmec cassette and regular mec and bla operons; β-lactamase production was confirmed by the nitrocefin test. The seesaw effect was striking (Fig. 1): in the daptomycin-nonsusceptible derivative, the penicillin MIC dropped to 0.125 μg/ml, in spite of persistent detection of the bla operon and of β-lactamase activity, and the cefoxitin MIC dropped to 2 μg/ml, despite persistent detection of the mecA gene and the mec operon in a type I SCCmec. Fig. 1. Diffusion tests using Etest strips (penicillin and cefoxitin). (A) Clinical isolate of daptomycin-susceptible S. haemolyticus (parent strain). (B) Daptomycin-nonsusceptible laboratory derivative. Although a number of theories have been advanced to account for the vancomycin/β-lactam and the daptomycin/β-lactam seesaw effect (7, 9), the underlying mechanisms remain poorly understood. Moreover, while previous hypotheses were essentially aimed at explaining a fall in methicillin resistance, thus largely pointing to some modulation of mecA expression, this case also involves a plunge in penicillin resistance, despite apparently normal—phenotypically and genotypically—β-lactamase production in the laboratory derivative. In other words, here the seesaw effect needs to be explained in light not only of the mec operon/PBP2a-mediated β-lactam resistance system but also of the bla operon/β-lactamase-mediated one. It is worth noting that, in a reported case of decreased susceptibility to daptomycin and vancomycin in S. aureus during prolonged therapy, a decreased penicillin MIC was found to be associated with the apparent loss of β-lactamase activity (5).

Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains

Multidrug‐resistant Klebsiella pneumoniae has become a relevant healthcare‐associated pathogen. Capsule, type 1 and 3 fimbriae (mrkA gene), type 2 quorum‐sensing system (luxS), synthesis of D‐galactan I (wbbM), LPS transport (wzm) and poly‐beta‐1,6‐N‐acetyl‐D‐glucosamine (pgaA) seem involved in K. pneumoniae biofilm. Nonenzymatic antibiotic resistance is related to nonexpression or mutation of porins (OmpK35 and OmpK36), and efflux pump (acrB) overexpression. The aim of this study was to analyse some virulence factors of K. pneumoniae isolates, and to evaluate possible correlations between their antibiotic resistance profile and ability to form biofilm.

Detection of hepatitis B virus DNA in serum using synthetic non-radioactive oligonucleotides.

A rapid and simplified technique for detecting hepatitis B virus (HBV) DNA by spot hybridisation in the sera of patients with different clinical forms of HBV infection was investigated using enzyme conjugated synthetic oligodeoxyribonucleotides as probes. These are able to hybridize to the S and C regions of the HBV L(-) DNA strand. When compared with a complete 32P-labelled HBV DNA probe, the synthetic oligonucleotides provided a sensitive and quick method for the routine survey of HBV infection. Moreover, the DNA extraction procedure used allowed the spot hybridisation technique to be applied and read easily and the results obtained within a few hours. It is concluded that synthetic cold probes can be used in hybridisation assays HBV DNA detection as part of current clinical laboratory procedures.