Persistence of TEM-52/TEM-92 and SHV-12 Extended-Spectrum β-Lactamases in Clinical Isolates of Enterobacteriaceae in Italy

Introduction

Strains producing extended-spectrum ß-lactamases (ESBLs) are able to hydrolyze expanded-spectrum cephalosporins and monobactams exhibiting often a multidrug-resistant phenotype, including resistance to aminoglycosides and fluoroquinolones. ⁴ It is known that the CTX-M-type β-lactamases have become among the most common and widespread ESBLs encountered in Enterobacteriaceae worldwide. ² Even though the CTX-M-type β-lactamases are playing a predominant role as emerging resistance determinants, TEM enzymes represent a good model as the evolution of antibiotic resistance in bacteria has occurred in a relatively short period. ¹⁵ As reported by Bush and Jacoby in their Web site (http://lahey.org/studies) TEM variants are 179 and >80% of them are ESBL. Classical ESBL have evolved from the broad-spectrum TEM-1/2 and SHV-type enzymes by amino acid substitutions in one or more positions.^1,7 The success of their evolution is due to their localization on transferable plasmids capable of rapid horizontal spreading among different enterobacterial species. ¹⁶ Some studies have been published in relation to the second Italian nationwide survey^3,5 on ESBLs, but no information has been given about the nature of TEM and SHV variants spreading in Italy. The goal of this study was to characterized TEM- and SHV-types recovered during the second Italian nationwide survey. ²

Materials and Methods

Results

In 2003, in Italy, a second nationwide survey of ESBLs production in Enterobacteriaceae was carried out. ³ The prevalence of ESBL-producing enterobacteria was evaluated both among inpatients and outpatients and the percentage of ESBL producers observed was 7.4% and 3.5%, respectively. TEM and/or SHV-type β-lactamase genes were detected in 453 of 583 (77.7%) ESBL producers. The presence of bla_TEM and bla_SHV genes was confirmed by PCR amplification carried out on these 453 isolates, including 158 Proteus mirabilis, 84 Escherichia coli, 71 Klebsiella pneumoniae, 42 Enterobacter aerogenes, 36 Providencia stuartii, 18 Klebsiella oxytoca, 15 Enterobacter cloacae, 12 Citrobacter freundii, 10 Serratia marcescens, 5 Citrobacter koseri, and 2 Morganella morganii. In 64 isolates the simultaneously presence of ESBLs with bla_TEM-1/2 and bla_SHV-1 or bla_SHV-11 genes was observed. By sequencing analysis, TEM-type ESBLs were confirmed in 154 isolates: P. mirabilis (101 isolates), P. stuartii (33 isolates), E. coli (11 isolates), E. aerogenes (2 isolates), M. morganii (2 isolates), C. koserii (2 isolates), K. pneumoniae (2 isolates), and S. marcescens (1 isolate), and included the following variants: TEM-5, TEM-6, TEM-12, TEM-15, TEM-24, TEM-26, TEM-29, TEM-52, TEM-92, TEM-134, and TEM-149. As shown in Table 1, TEM-92 (in 84 isolates) and TEM-52 (in 38 isolates), restricted to P. mirabilis, P. stuartii, and M. morganii, were the most common variants being found in 79% of total TEM-type producers.

The nature of bla_SHV variants was investigated in 74 SHV-type-ESBL-producing enterobacterial isolates: E. coli (15 isolates), K. pneumoniae (25 isolates), K. oxytoca (7 isolates), C. freundii (6 isolates), E. cloacae (6 isolates), E. aerogenes (12 isolates), S. marcescens (3 isolates), and included the following variants: SHV-2a, SHV-5, SHV-12, and SHV-28. The most diffused variant was SHV-12 (Table 2).

Discussion

ESBL-producing bacteria among hospitalized and nonhospitalized patients represent a serious problem worldwide. In the last two decades, two nationwide survey were undertaken in Italy on the spreading of ESBL-producing Enterobacteriaceae.^2,17 Results of the present investigation revealed that both TEM- and SHV-type ESBLs are widespread in all Italian hospitals involved in the study with the more diffused TEM-92/TEM-52 and SHV-12. Overall, TEM-52/92 enzymes were identified in 122 of 154 isolates (79.2%). The most interesting mutation found in TEM-52/92 is M182T, which has the ability to reverse the destabilizing effects of antibiotic or inhibitor-resistant mutations. ⁶ The M182T mutation in combination with E104K and G238S substitutions in TEM-52/92 enzyme increases the cefotaxime affinity. Kinetic data reported for TEM-92 ⁸ showed that this enzyme was able to efficiently hydrolyzed several β-lactams including penicillins, narrow-spectrum cephalosporins, and cefotaxime. Ceftazidime was hydrolyzed somewhat less efficiently while no hydrolysis was detectable with aztreonam. TEM-92 enzyme differs to TEM-52 only for Q6K mutation in the signal peptide sequence. ⁸ This substitution could have an important role in efficient protein secretion across the membrane. Two new variants also appeared for the first time in Italy: TEM-134 ¹¹ and TEM-149. ¹⁰ Among TEM-derived β-lactamases 11 different variants have been found (TEM-5, TEM-6, TEM-12, TEM-15, TEM-24, TEM-26, TEM-52, TEM-92, TEM-134, and TEM-149). It should be noted that in eight variants (TEM-5, TEM-6, TEM-12, TEM-24, TEM-26, TEM-29, TEM-134, and TEM-149) is present the substitution at position 164 where an arginine residue is changed in serine (TEM-5, TEM-12, TEM-24, TEM-26, and TEM-149) or histidine (TEM-6, TEM-29, and TEM-134). Normally, the side chain of residue Arg164 is involved in stabilizing electrostatic and hydrogen bond interaction with the side chain of Asp179. On the basis of the modeling and activity studies, the loss of the electrostatic interaction with Asp179 as the result of a serine or hystidine substitution at position 164 destabilizes the omega loop structure.^12–14 This effect leads to easier access of the active site for the bulky extended-spectrum antibiotics (cefotaxime, ceftazidime, and aztreonam).

In the present study we showed a persistence of SHV-5 and SHV-12 enzymes in all participating hospitals. In SHV enzymes the serine substitution at position 238 as in SHV-2a, SHV-5, and SHV-12 enzymes results in a greater increase of cefotaxime hydrolysis by the SHV enzyme than by TEM enzymes. Minor number of extended-spectrum substitutions are observed in SHV natural variants respect to TEM enzymes. It should be noted that substitution at position 164 is not been seen in any SHV-variants. In fact the high level of hydrolytic activity for cefotaxime and ceftazidime in SHV enzymes is only due to E240K and G238S substitutions.

In the last decades, no new β-lactam derivatives have been introduced in the clinical practice apart the new antipneumococcal cephalosporins developed to overcome the altered PBP in Gram-positive and anti-MRSA bacteria. Until now, carbapenems have been considered as drug of last line against enterobacteria producing ESBLs. Moreover, the prolonged use of these molecules could contribute to the emergence not only of new metallo-β-lactamases (i.e., NDM1 enzyme) but also of class A carbapenemases (i.e., KPC-types, GES-types, SME-types, and SHV-38).^18,19 able to acquire the more powerful hydrolytic activity versus these antibiotics by new point mutation.