**5.3. Results and discussion**

The major limit of the present study is the scarcity of carbapenemases of environmental source deposited in the public databases. Most of environmental beta-lactamases found belong to Classes B and D. The archaeal beta-lactamases found are metallo-hydrolase, and metal ions probably contribute to enzymes stability in extreme environmental conditions. Searching was extended to Eukaryota domain of life, but any sequences were included in the present study. Only two beta-lactamases-like of eukaryotic origin were retrieved—*Homo sapiens* (NP\_057111) and *Caenorhabditis elegans* (NP\_497107). Even they show MBL-folding, their similarity with other class B carbapenemases are very low, being more close to one hypothetical protein from *Sulfolobus acidocaldarius* (WP\_01538554)—20.5% identity with NP\_057111; 23.2% with NP\_497107, respectively.

On the other hand, environmental Classes A, B, and D carbapenem-hydrolyzing enzymes are very similar with carbapenemases of clinical sources. Some carbapenemases, such as IMI-2, IMI-3, NDM-1, or OXA-48, are the same regardless their origin as observed by sequences pairwise comparison [50, 51]. For further statistical analysis, identity percent was recovered. Student's *t*-test assuming unequal sample variance was performed. The limit of this approach is due to the differences in the sequences length. The Needle program calculates identity, similarity, and score according to number of residues. Anyway, observing the data from **Table 4**, we noticed that environmental serine beta-lactamases are more similar with their clinical enterobacterial counterparts than with nonfermenters carbapenemases. The most striking differences are observed for subgroups IMI-3 of uncultured bacteria (ALJ52278) isolated from river sediment in China and OXA-48-like of uncultured bacteria (AJF40233) isolated from river sediment in Portugal. For ALJ52278 (IMI-3) beta-lactamase, 73.03% identity mean (CI 95 ± 25.33) with Class A carbapenemases of clinical *Enterobacteria* was noticed. More, sequences pairwise comparison reveals 93.50% identity with subgroups IMI-2 (*Enterobacter asburidae* WP\_032491237), IMI-2 (*E. coli* AEU10762), and IMI-14 (*Enterobacter hormaechei*


APY16311) (data not shown). The Class D carbapenemase AJF40233 (OXA-48-like) displays 80.35% identity mean (CI 95 ± 34.61) with its clinical *Enterobacteria* counterparts. Sequences pairwise comparison shows 91.00% identity with OXA-48-like (*Klebsiella pneumoniae* 5FAT and *K. pneumoniae* 4WMC) and 91.70% identity with OXA-244 (*K. pneumoniae* AGC60012) (data not shown). In contrast, for Class B carbapenemases, there are no notable differences when comparing environmental carbapenemases with those of clinical *Enterobacteria* or nonfermenters of Gram-negative bacilli origin. Particularly, all three NDM-1 carbapenemases of environmental sources included in the present study (**Table 3**) are identical with the carbapenemases isolated from *K. pneumoniae* (3SPU), *Enterobacteria*, or from the nonfermenter *Pseudomonas aeruginosa* (ALU10771). This is the most obvious illustration of long-term consequences of antibiotics use in livestock farms. Contrary, as it is shown in **Table 4**, Archaeal

**Class Source Organism (accession number) isolation source Beta-lactamase Residues**

E *Klebsiella pneumoniae* (5FAT) Oxa-48 complex with

*Klebsiella pneumoniae* (4WMC) Oxa-48 complex with

N *Pseudomonas aeruginosa* (AAQ76282) OXA-50 262

*Escherichia coli* (3QNC) OXA-10 244 *Klebsiella pneumoniae* (AGC60012) OXA-244 265

*Acinetobacter baumannii* (ADB28891) OXA-160 275 *Acinetobacter baumannii* (4WM9) OXA-24 245 *Acinetobacter radioresistens* (ACE63186) OXA-23 273 *Acinetobacter baylyi* (ACH99101.1) OXA-72 275

OXA-48 like 215

http://dx.doi.org/10.5772/intechopen.69630

OXA-48 like 190

OXA-181 173

OXA-48-like 248

243

67

242

Fpi-1602

The Emerging Problems of Carbapenem-Resistant Gram-Negative Bacillary Pneumonia

Avibactam

D ES *Escherichia coli* (AJA05008)/*Halimione portulacoides*,

*portulacoides*, Portugal

*portulacoides*, Portugal

*Citrobacter freundii* (AJA05009)/*Halimione* 

*Pantoea eucalypti (*AJA05006*)*/*Halimione* 

Uncultured bacterium (AJF40233)/river water,

Portugal

Portugal

**Table 3.** The main characteristics of beta-lactamase sequences.

Multiple sequence alignment of Classes A and D carbapenemases demonstrate that the activesite residues are very well conserved. Class B carbapenems, which mediate resistance to all betalactamases except aztreonam, are particularly interesting; they are found in bacteria, Archaea, and similar proteins, even in eukaryotes. Further, multiple sequence alignment (**Figure 10**)

metallo-hydrolases show low similarities with other Class B carbapenemases.


**Table 3.** The main characteristics of beta-lactamase sequences.

**Class Source Organism (accession number) isolation source Beta-lactamase Residues**

*Sulfolobus acidocaldarius* (WP\_015385554) Hypothetical protein 298 *Hadesarchaea* archaeon (KUO42968) MBL fold metallo-hydrolase 395 *Archaeoglobus fulgidus* (WP\_010877604) MBL fold metallo-hydrolase 293 *Vulcanisaeta distributa* (WP\_013336995) MBL fold metallo-hydrolase 306

IMI-3 280

NDM\_FIM-like\_MBL-B1 270

NDM-1 (plasmid) 270

NDM-1 (plasmid) 270

227

MBL-fold ES *Sulfolobus acidocaldarius* (WP\_011278945) MBL fold metallo-hydrolase 305

A ES *Enterobacter asburiae (*AIL29239)/water IMI-2 192

E *Escherichia coli* (WP\_015059046) GES-20 287

N *Pseudomonas aeruginosa* (WP\_021018677) KPC 293

E *Klebsiella pneumoniae* (4UWS) VIM-26 266

N *Pseudomonas aeruginosa* (4NQ2) VIM-2 261

*Aeromonas hydrophila* (1X8I) CphA complex with

*Klebsiella pneumoniae* (5A87) VIM-5 248 *Klebsiella pneumoniae* (3SPU) NMD-1 265

*Pseudomonas aeruginosa* (AAR15341) SPM-1 276 *Pseudomonas aeruginosa* (ALU10771) NMD-1 270 *Pseudomonas aeruginosa* (AGH20684) IMP-15 246

Biapenem

*Pseudomonas aeruginosa* (4GNU) GES-5 287 *Acinetobacter baumannii* (3TSG) GES-14 287

*Serratia fonticola* (4EV4) SFC-1 E166A mutant 283 *Serratia marcescens* (1DY6) SME-1 267 *Enterobacter asburiae* (WP\_032491237) IMI-2 292 *Escherichia coli* (AEU10762) IMI-2 292 *Enterobacter hormaechei* (APY16311) IMI-14 292

uncultured bacterium (ALJ52278)/river sediment,

*Acinetobacter junii* (YP\_009062718)/livestock farms,

*Acinetobacter calcoaceticus* (YP\_009060354)/

Haithe River, China

B ES *Serratia* sp. Sr\_CGKV333\_2014 (ALL98459)/ poultry flock soil, India

livestock farms, China

China

**Archaea**

66 Contemporary Topics of Pneumonia

**Bacteria**

APY16311) (data not shown). The Class D carbapenemase AJF40233 (OXA-48-like) displays 80.35% identity mean (CI 95 ± 34.61) with its clinical *Enterobacteria* counterparts. Sequences pairwise comparison shows 91.00% identity with OXA-48-like (*Klebsiella pneumoniae* 5FAT and *K. pneumoniae* 4WMC) and 91.70% identity with OXA-244 (*K. pneumoniae* AGC60012) (data not shown). In contrast, for Class B carbapenemases, there are no notable differences when comparing environmental carbapenemases with those of clinical *Enterobacteria* or nonfermenters of Gram-negative bacilli origin. Particularly, all three NDM-1 carbapenemases of environmental sources included in the present study (**Table 3**) are identical with the carbapenemases isolated from *K. pneumoniae* (3SPU), *Enterobacteria*, or from the nonfermenter *Pseudomonas aeruginosa* (ALU10771). This is the most obvious illustration of long-term consequences of antibiotics use in livestock farms. Contrary, as it is shown in **Table 4**, Archaeal metallo-hydrolases show low similarities with other Class B carbapenemases.

Multiple sequence alignment of Classes A and D carbapenemases demonstrate that the activesite residues are very well conserved. Class B carbapenems, which mediate resistance to all betalactamases except aztreonam, are particularly interesting; they are found in bacteria, Archaea, and similar proteins, even in eukaryotes. Further, multiple sequence alignment (**Figure 10**)


**6. Final remarks**

for changing the scent [52].

Carbapenems remain a valid option for the treatment of ESBL *Enterobacteriaceae* pneumonia. Prolonged treatments with beta-lactamine associated with other co-morbidities of the

**Figure 10.** Multiple sequence alignment of Class B beta-lactamase. The cysteine residues involved in the second Zn2+ binding, highly conserved in bacteria, are written in white on black background; the residues noticed in Archaea are

The Emerging Problems of Carbapenem-Resistant Gram-Negative Bacillary Pneumonia

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69

highlighted - the aspartic acid are underlined and the cysteine are written on grey background.

Essential oils have, no doubt, beneficial effects, but some providers excessively claim many possible effects—from antibacterial activity to neurological benefits—not always consistent with reality. Moreover, nowadays, there exists all sort of mixtures, and we can only hope that the ingredients are chosen following logical connections between their activities. Herein, these problems were not debated, but it is not a trivial question about the validity of all plant extracts. Just notice that the only mixture tested (an inhalant) proved no antibacterial activity, at least against *A. baumannii* and *E. coli* strains. On the other hand, tyme-clove association proved to be beneficial in Gram-negative bacilli respiratory infections. The oregano EO has an excellent antibacterial activity, but its effect is antagonized by negril EO. Other plant extracts could be added for their anti-inflammatory effect or just

One important question arises from here—what else, apart from widely using of antibiotics, could influence the persistence of antibiotic genes in hospital facilities? Industrial wastes could remain for a long period of time in the environment, notably in groundwater [8]. Carbapenemases from hospital sources are, no doubt, the major factor in the evolution of these enzymes, hospital residues being, definitely, a source of wastewater pollution. Further, the carbapenemases produced by natural environmental bacteria and Archaea significantly contribute to selection of new mutations. Soil bacterial species greatly influences our life; therefore, a genome-scale metabolic network [53] has proved to be a valid approach to evaluate the complex dynamics of soil bacterial species, mostly in geographic areas near huge hospitals with many departments. Also, innate resistance to antibiotics has raised over time a

growing interest for a rational design of new antibacterial compounds [54].

patients, rapidly changed the phenotypic pattern of resistance.

**Table 4.** Pairwise comparison of beta-lactamase sequences of environmental sources with *Enterobacteria* and nonfermenters carbapenemases.

highlights the notable differences between Archaea and bacteria. Residues involved in Zn2+ ions are very well conserved with the exception of Cys221, which is replaced by Asp in Archaea. However, nearby Cys residues were noticed. Since their crystal structures are not solved, we can just assume their involvement in metal ion binding.

The observation that Archaea contain different beta-lactamases demonstrates that human behavior has not profoundly altered natural environment. Or some microorganism communities have regulatory mechanisms so flexible that rapidly adapt at new environmental factors. For example, *Acinetobacter* is no longer considered just a free-living organism found in soil, water, and skin of human and warm-blooded animals, but an important multidrug-resistant pathogen.


**Figure 10.** Multiple sequence alignment of Class B beta-lactamase. The cysteine residues involved in the second Zn2+ binding, highly conserved in bacteria, are written in white on black background; the residues noticed in Archaea are highlighted - the aspartic acid are underlined and the cysteine are written on grey background.
