Emergence of Carbapenem-resistant Clinical Isolates of Providencia Species

Providencia is a genus of Gram-negative and non-spore forming bacteria belonging to the family Morganellaceae, which causes opportunistic infections in humans. Of the 10 Providencia species identified to date, three, P. alcalifaciens, P. rettgeri and P. stuartii, are clinically important. P. alcalifaciens causes diarrhea, including outbreaks arising from food-borne infections, and P. stuartii and P. rettgeri have been found to cause hospital acquired urinary tract infections. Four isolates of P. rettgeri and one isolate of P. stuartii were obtained from urine samples of five patients in Japan in 2018. All five isolates were highly resistant to carbapenems. Three isolates harbored blaIMP-70, encoding a variant of IMP-1 metallo-β-lactamase, with two amino acid substitutions (Val67Phe and Phe87Val), one isolate harbored two copies of blaIMP-1 and one isolate harbored blaIMP-11. Expression of blaIMP-70 conferred carbapenem resistance in Escherichia coli. Recombinant IMP-10, an IMP-1 variant with Val67Phe but without Phe87Val, had significant higher hydrolytic activities against meropenem than recombinant IMP-1, indicating that the Val67Phe amino acid substitution alters activities against meropenem in IMP-70. These results suggest that Providencia species. become more highly resistant to carbapenems by acquisition of two copies of blaIMP-1 or by mutations in blaIMP that result in amino acid substitutions, such as blaIMP-70.


Providencia species as human pathogens
A study of the enteropathogenicity of P. alcalifaciens isolated from a child and two adults with diarrhea demonstrated that this species causes diarrhea in humans by invading the intestinal mucosal epithelium 7) .P. alcalifaciens was subsequently isolated from 2.1% of the stool specimens of diarrheal children younger than 5 years of age, indicating that this organism is significantly associated with diarrhea 8) .A large outbreak of food-borne infection caused by P. alcalifaciens occurred among children and teachers at two kindergartens and one high school in November 1996 in Fukui, Japan 9) .Specifically, of the 610 children and teachers who ate lunch cooked at a single catering facility, 270 showed symptoms of gastroenteritis 9) .Recent outbreaks of P. alcalifaciens have indicated that infection with this organism is a public health concern in both developing and developed countries 10) .Although epidemiological studies suggest that P. alcalifaciens causes diarrhea by invading the intestinal mucosa 10) , the pathogenesis of P. alcalifaciens has not been established at the molecular level.
P. stuartii and P. rettgeri have been found to cause hospital acquired urinary tract infections 11) and have been shown to be the most common causes of urinary tract infections in hospitalized patients.
In addition, P. stuartii and P. rettgeri have been found to cause pneumonia, meningitis, endocarditis, wound infections and bloodstream infections 11) , and P. stuartii was found to cause invasive endocarditis 12) and neonatal sepsis 13) .P. alcalifaciens, P. rettgeri and P. stuartii were isolated from 17.6% of stool samples of patients with diarrhea at the Kansai airport quarantine station in 2002, with vomiting being especially frequent in patients infected with P. rettgeri, indicating that these three Providencia species cause travelers' diarrhea 5) .

Emergence of carbapenem-resistant Providencia species
The emergence and spread of carbapenem-resistant Gram-negative pathogens have become serious public health problems worldwide 14) .Most of these carbapenem-resistant isolates produce metallo-βlactamases (MBLs), including IMP-, NDM-and VIM-type MBLs 14) , which confer high resistance against all β-lactams (penicillins, cephalosporines and carbapenems) except for monobactams 15) .Clinical isolates of carbapenem-resistant P. rettgeri producing IMP-1 MBL were first identified by laboratory-based surveillance in the Kinki region of Japan in 2000 16) .Clinical isolates of P. stuartii producing VIM-19 MBL were first identified in 2008 in Algeria 17) .To date, there have been 16 Figure 1 Maximum-likelihood (ML) tree based on single nucleotide polymorphisms (SNPs) in the core genome among contigs of strains, showing the relationships among type strains of the genus Providencia.Bootstrap values, expressed as percentages of 1,000 replications, are shown at the branching points when >50 %. reports of carbapenem-resistant P. rettgeri, eight of carbapenem-resistant P. stuartii and one of carbapenem-resistant P. vermicola (Table 1).Most of these were clinical isolates, but one was obtained from a hospital environment and one from pet turtles (Table 1).
All of these isolates produced MBLs, with the majority of carbapenem-resistant P. rettgeri isolates producing IMP-type or NDM-type MBLs (Table 1).IMP-type MBLs were detected in isolates from Japan, Korea, and the United States, whereas NDMtype MBLs were detected in isolates worldwide.We obtained four clinical isolates of carbapenemresistant P. rettgeri, which produced IMP-1, IMP-11 or IMP-70.One IMP-1 producing isolate was from Saitama, Japan, one IMP-11 producing isolate was from Kochi, Japan, and two IMP-70 producing isolates were from Osaka, Japan 18) .
Most of the carbapenem-resistant P. stuartii isolates, obtained in Algeria, Greece and Korea, produced VIM-type MBLs.Carbapenem-resistant P. stuartii isolates producing NDM-type MBLproducing P. stuartii were obtained in Afghanistan and Peru, and we described an IMP-type MBLproducing P. stuartii from Japan 18) .Carbapenemresistant P. vermicola isolates producing NDM-1 were isolated in the Congo.

Carbapenem-resistant clinical isolates of Providencia species in Japan
We obtained four clinical isolates of P. rettgeri and one clinical isolate of P. stuartii from the urine a Iwata S, Tada T, Hishinuma T, et al: Antimicrob Agents Chemother, 2020; 64. 18) dash (-) indicates there was no information about the location of MBL-encoding genes, Inc type and isolation source.
One isolate harbored aminoglycoside-and carbapenem-resistant genes on the chromosome, whereas the other four harbored these genes on plasmids.

Carbepenemase activities of IMP-1 MBL variants
All five P. rettgeri and P. stuartii clinical isolates were resistant to imipenem and meropenem, and three, two P. rettgeri isolates and one P. stuartii isolate, were highly resistant to both carbapenems, with minimum inhibitory concentrations (MICs) of 512 μg/ml (Table 3).These three highly carbapenem-resistant isolates harbored bla IMP-70 , whereas, of the other two, one harbored bla IMP-1 and the other harbored bla IMP-11 .IMP-70 is a variant of IMP-1 with two amino acid substitutions, Val67Phe and Phe87Val; IMP-10 is a variant of IMP-1 with one amino acid substitution, Val67Phe; and IMP-1(F87V) is a variant of IMP-1 with one amino acid substitution, Phe87Val.E. coli expressing bla IMP-1 , bla IMP-10 , bla IMP-1(Phe87Val) , and bla IMP-70 showed significantly higher MICs for all carbapenems tested than a vector control (Table 4).The MICs for all carbapenems of the vector control ranged from ≤0.06 to 0.125.E. coli expressing bla IMP-70 showed higher MICs for doripenem and meropenem, but the same MICs for imipenem and panipenem, than E. coli expressing bla IMP-1 .E. coli expressing bla IMP-10 showed a significantly higher MIC for doripenem and an increased MIC for meropenem.Assessment of the carbepenemase activities of recombinant IMP-1, IMP-10, IMP-1(Phe87Val) and IMP-70 showed that IMP-10 had greater hydrolytic activities than IMP-1 against meropenem, with the k cat / K m values of IMP-70 and IMP-10 being 2.3-and 3.4-fold higher, respectively, than those of IMP-1 (Table 5).In contrast IMP-70 and IMP-1 showed similar carbapenemase activities against doripenem, imipenem and panipenem, and IMP-1(Phe87Val) showed similar or reduced carbapenemase activities against all carbapenems tested.These results suggest that, in IMP-70, the Val67Phe amino acid substitution, but not the Phe87Val substitution, is important for the significantly increased carbapenemase activity against meropenem.The Val67 in IMP-1 is located at the end of "loop1", close to the active site consisting of amino acids residues 60 to 66 (Figure 2) 19) .Loop1 is a major determinant for the tight binding of substrates in the active site 19) .A Val67Phe amino acid substitution in IMP-43, a variant of IMP-7, has been reported to increase catalytic activities against imipenem and meropenem 20) .Amino acid substitutions at residue 67 in IMP-1 MBLs affect their hydrolytic activity against β-lactams 21) .Residue 67 was reported to be important for substrate binding in VIM-type MBLs 22) .Residue 87 plays a crucial role in the stability of VIM-2 23) .IMP-44, a variant of IMP-11 with two substitutions (Val67Phe and   Phe87Ser), had more efficient catalytic activities against carbapenems than those of IMP-11 24) .These results suggest that co-occurrence of two amino acid substitutions at these two positions increase the enzymatic activities of IMP-44, whereas the Phe87Val substitution did not affect the enzymatic activities of IMP-70.The substitution of Phe87 by a polar amino acid such as Ser, but not by a hydrophobic amino acid such as Val, may affect enzymatic activities.

Biological significance of two copies of blaIMP-1 in tandem
One of the P. rettgeri isolates was found to harbor two copies of bla IMP-1 , in tandem on the chromosome, consisting of a repeat of the genetic structure int1Δ-bla IMP-70 -qacEΔ1-sul1 (Figure 3).To confirm the presence of the two copies of bla IMP-1 , sequences were amplified by PCR using a primer set targeting the two copies.Amplification resulted in a 3.5-kbp PCR product as expected based on the whole-genome sequence, indicating that this isolate of P. rettgeri harbored two tandem copies of bla IMP-1 on the chromosome.Western blotting analysis revealed that all five isolates tested produced IMPtype MBLs (Figure 4).Of these five isolates, the P. rettgeri isolates harboring two copies of bla IMP-1 produced the largest quantities of IMP-type MBL (Figure 4), indicating these two copies of bla IMP-1 produce high amounts of IMP-1 MBL.

Conclusions
The genus Providencia, belonging to the family Morganellaceae, consists of 10 species.Of these, three species, P. alcalifaciens, P. rettgeri and P. stuartii, are clinically important.P. alcalifaciens causes diarrhea by invading the intestinal mucosa, whereas P. stuartii and P. rettgeri have been found to cause hospital acquired urinary tract infections, as well as pneumonia, meningitis, endocarditis, wound infections, bloodstream infections, and travelers' diarrhea.Clinical isolates of carbapenemresistant P. rettgeri producing IMP-1 MBL were first identified during laboratory-based surveillance in 2000 in Japan.To date, there have been 16 reports of carbapenem-resistant P. rettgeri, eight of carbapenem-resistant P. stuartii and one of carbapenem-resistant P. vermicola, with most of these being clinical isolates.
We recently obtained four P. rettgeri isolates and one P. stuartii isolate from urine samples of five patients.All five isolates were highly resistant to carbapenems.Three isolates harbored bla IMP-70 , encoding a variant of IMP-1 MBL with two amino acid substitutions, and one each harbored bla IMP-1 and bla IMP-11 .Molecular analyses of these isolates strongly suggest that Providencia species become more highly resistant to carbapenems by acquisition of two copies of bla IMP-1 or by mutations in bla IMP genes that result in amino acid substitutions, such as bla IMP-70 .

Figure 3
Figure 3 Genomic environments of bla IMP-1 and bla IMP-70 in clinical isolates of P. rettgeri and P. stuartii.Genes are represented as arrows, which indicate their transcription orientations and relative lengths.MBL genes, tnp genes, and truncated genes are shown as black arrows, gray arrows, and Δ, respectively.Label orf1 represents a gene encoding a hypothetical protein, and orf2 represents a gene encoding an ATP-binding protein.This figure is a modified version of FIG 1 in reference 18. (Iwata S, Tada T, Hishinuma T, et al: Emergence of Carbapenem-Resistant Providencia rettgeri and.Antimicrob Agents Chemother, 2020; 64.)

Table 1
Reports of P. rettgeri, P. stuartii and P. vermicola producing MBL a

Table 2
Genetic characterization of carbapenem-resistant Providencia species isolates

Table 3
Drug susceptibility profiles of Providencia species clinical isolates

Table 4
Drug susceptibility profiles of E. coli expressing IMP-1, IMP-10, a variant of IMP-1 with an amino acid substitution

Table 5
18)etic parameters of β-lactamases IMP-1, IMP-10, a variant of IMP-1 with an amino acid substitution (F87V) and IMP-70 with substrates a Iwata S, Tada T, Hishinuma T, et al: Antimicrob Agents Chemother, 2020; 64.18)b Km and kcat were calculated as means ± SD from three independent experiments. a