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  Table of Contents  
Year : 2011  |  Volume : 29  |  Issue : 3  |  Page : 305-308

CTX-M-9 group extended-spectrum β-lactamases in neonatal stool isolates: Emergence in India

1 Division of Bacteriology, National Institute of Cholera and Enteric Diseases, P33, CIT Road, Scheme XM, Beliaghata, Kolkata - 700 010, India
2 Department of Neonatology, Institute of Postgraduate Medical Education and Research, SSKM Hospital, Kolkata - 700020, India

Date of Submission09-Dec-2010
Date of Acceptance01-Jul-2011
Date of Web Publication17-Aug-2011

Correspondence Address:
S Basu
Division of Bacteriology, National Institute of Cholera and Enteric Diseases, P33, CIT Road, Scheme XM, Beliaghata, Kolkata - 700 010
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.83919

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 ~ Abstract 

The study reports for the first time the identification of CTX-M-14-like and CTX-M-27-like extended-spectrum β-lactamases (ESBLs) belonging to the CTX-M-9 group in Klebsiella pneumoniae and Escherichia coli isolated from the neonatal stool in India. The plasmid carrying the blaCTX-M-9 group in both the isolates was transferable. Till date, no other CTX-M group, except the CTX-M-1 group, has been reported from India. A total of 77% of the neonates had ESBL-producing K. pneumoniae or E. coli in their stool, and blaCTX-M-15 was the predominant ESBL gene. Although the CTX-M-9 group was found in the stool and did not cause infection, the detection of the CTX-M-9 group might be a prelude to future infections.

Keywords: CTX-M-9 group, CTX-M-14-like, CTX-M-27-like, India, neonatal sepsis

How to cite this article:
Roy S, Mukherjee S, Singh A K, Basu S. CTX-M-9 group extended-spectrum β-lactamases in neonatal stool isolates: Emergence in India. Indian J Med Microbiol 2011;29:305-8

How to cite this URL:
Roy S, Mukherjee S, Singh A K, Basu S. CTX-M-9 group extended-spectrum β-lactamases in neonatal stool isolates: Emergence in India. Indian J Med Microbiol [serial online] 2011 [cited 2021 Jan 15];29:305-8. Available from:

 ~ Introduction Top

Sepsis remains a major cause of neonatal deaths in developing countries, including India. [1] A compounding factor in the treatment of sepsis is the emergence of extended-spectrum b-lactamases (ESBLs) producing Enterobacteriaceae. [2] CTX-Ms are b-lactamases that are extremely diverse and have now been disseminated around the globe. [3] This communication reports the presence of CTX-M-9 group enzymes in Klebsiella pneumoniae and  Escherichia More Details coli isolated from the neonatal stool. To the best of our knowledge, this is the first report from India about the presence of the blaCTX-M-9 group producing Enterobacteriaceae, as earlier studies have reported only blaCTX-M-15 belonging to the CTX-M-1 group. [4] This is an indication that new mechanisms of resistance might be emerging in India.

An important part of infection prevention is monitoring infection. Monitoring the gut for antibiotic-resistant organisms in the Intensive Care Unit (ICU) is an effective way of assessing the change in the antibiotic resistance patterns of the organisms. We have evaluated the stool samples of neonates in the Neonatal Intensive Care Unit (NICU).

 ~ Materials and Methods Top

The study was carried out in a 20-bed NICU in a tertiary care center in Kolkata, India. Stool samples of 210 neonates, whose parents consented to the study, were surveyed during 2007. The samples were streaked onto MacConkey agar and the isolates were identified by using the ID 32 E kit (bioMe΄rieux, Marcy l'Etoile, France). Antibiotic (BD Diagnostics, Sparks, MD, USA) susceptibility test was carried out as per Clinical and Laboratory Standards Institute (CLSI) criteria. [5] The antibiotics (μg) [Table 1] were selected depending not on the use in neonates, but rather on the spectrum of activity against potentially multidrug-resistant organisms. The minimum inhibitory concentration (MIC) values (μg/ml) for cephalosporins were determined using E-tests (AB Biodisk, Solna, Sweden). ESBL production was confirmed by the cephalosporin/clavulanic acid combination disc test. [5] The genotypic analysis for the blaTEM, blaSHV, blaOXA-1, and blaCTX-M genes was performed. [6] Isolates were screened for the presence of class 1, 2, and 3 integrons. Sequencing of blaCTX-M was carried out by using the CTX-M-1 and CTX-M-9 group primers and was analysed with the help of the Basic Local Alignment Search Tool (BLAST) program ( Pulsed-field gel electrophoresis (PFGE) was performed in a CHEF-DR III system (Bio-Rad Laboratories, Hercules, CA, USA) with XbaI restriction enzymes ( The presence of plasmids and their approximate sizes were estimated using logarithmic plots generated with plasmids of known molecular mass. The conjugal transfer of blaCTX-M to the sodium azide-resistant recipient E. coli J53 (kind gift of George A. Jacoby, Lahey Clinic, Burlington, MA, USA) by a broth mating assay using Luria-Bertani agar plates containing cefotaxime (4 μg/ml) and sodium azide (100 μg/ml) was also attempted.
Table 1: Antibiotic susceptibility profi le, MIC measurements, detection of ƒÒ-lactamase genes, and sequencing of blaCTX-M of three strains isolated from two neonates

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 ~ Results Top

On survey, K. pneumoniae and E. coli isolates from 77% (162/210) of the stool samples showed phenotypic evidence of ESBL production. ESBL-positive isolates were then screened by gene-specific primers and were CTX-M-1 group positive, except for two cases which were CTX-M-9-group positive. The description of the cases and the isolates harbouring CTX-M-9 group is presented here.

The first isolate, K. pneumoniae (I-1), possessing CTX-M-9 group was from the stool of one of the neonates (case 1). The patient was a term (37 weeks), male baby, weighing 2830 g, delivered by caesarean section. On the first day of life, the baby did not feed well, was lethargic, presented with severe apnoea, bradycardia, and hypothermia. Antibiotics were started after blood was drawn. K. pneumoniae (I-2) was isolated from the blood of the baby. PFGE patterns of K. pneumoniae isolated from the stool (I-1) and blood (I-2) of the baby with culture-proven sepsis were distinct [Figure 1].
Figure 1: Pulsed-fi eld gel electrophoresis (PFGE) of XbaI-digested genomic DNA of the CTX-M-1 group and CTX-M-9 group producing Klebsiella pneumoniae Scientific Name Search  isolated from blood (I-2) and stool (I-1), respectively. Lane 1:  Salmonella More Details serotype Braenderup H9812 as reference standard (band sizes in kilobases). Lane 2: CTX-M-15 producing Klebsiella pneumoniae (I-2). Lane 3: CTX-M-14 producing Klebsiella pneumoniae (I-1)

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The second isolate, E. coli (I-3), was from the stool of a term (37 weeks), male baby (case 2), weighing 3350 g, and delivered vaginally. On the first day of life, the baby was lethargic and hypothermic. The baby had clinical sepsis; however, the blood culture result was negative. Both the neonates were given amikacin and gentamicin. They improved and were later discharged.

All three isolates (two from the stool, I-1 and I-3; one from blood, I-2) were multidrug-resistant. MIC (μg/ml) values for these isolates showed similar resistance profiles [Table 1]. All three isolates possessed blaSHV and blaTEM, along with blaCTX-M [Table 1]. I-2 showed the presence of CTX-M-15 belonging to the CTX-M-1 group, but the two stool isolates (I-1 and I-3) showed the presence of blaCTX-M-14-like and blaCTX-M-27-like genes, respectively [Table 1], belonging to CTX-M-9 group. The accession numbers of the submitted blaCTX-M-14-like and blaCTX-M-27-like genes are AB545871 and AB545872, respectively. Integron 1 was detected only in the blood isolate (I-2), but no integrons could be identified in the stool isolates (I-1 and I-3). Two large plasmids of approximately >212 kb were identified in I-1. I-3 possessed three large plasmids, one of approximately 212 kb and the two others of approximately >212 kb. Plasmids carrying the blaCTX-M-9 group were found to be transferable. The presence of blaCTX-M-9 group was confirmed by polymerase chain reaction (PCR) in the transconjugants and plasmids transferred by conjugation, as shown in [Figure 2].
Figure 2: Plasmid profiles of one isolate of Klebsiella pneumoniae (I- 1) (Lane 2) and  Escherichia coli Scientific Name Search ane 4) harbouring blaCTX-M-14-like and blaCTX-M-27-like, respectively, isolated from the stool of neonates,
and the blaCTX-M-9 group containing transconjugants of Klebsiella pneumoniae (Lane 1) and Escherichia coli (Lane 3). Lanes 5 and 6: Shigella fl exneri YSH 6000 and Escherichia coli K12 V517 were used as a guide to molecular size, respectively

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 ~ Discussion Top

CTX-M enzymes are a family of plasmid-mediated ESBLs that preferentially hydrolyse cefotaxime and are not closely related to the TEM and SHV enzymes. The description of the first CTX-M-type ESBL, namely, CTX-M-1, goes back to the late 1980s. Thereafter, new variants have been increasingly reported. At present, 111 variants have been assigned in the Lahey clinic database ( They are clustered into five lineages or subgroups (CTX-M-1 group, CTX-M-2 group, CTX-M-8 group, CTX-M-9 group, and the CTX-M-25 group) according to the amino acid sequence similarities. [3] The members of each group share >94% identity, whereas ≤90% identity is observed between the members belonging to distinct groups. [3] CTX-M-15, belonging to the CTX-M group 1, is the most widely distributed CTX-M enzyme globally and is also the most prevalent CTX-M enzyme in India. [4] The other CTX-M enzymes reported from India are CTX-M-3 and CTX-M-28. [7],[8] All the three enzymes (CTX-M-15, -3, and -28) belong to the CTX-M group 1. In addition, as far as newborns are concerned, there is only one report on the CTX-M group 1 from India. [9] To the best of our knowledge, the present study is the first report of the isolation and characterisation of blaCTX-M-14-like and blaCTX-M-27-like genes, belonging to CTX-M group 9, from India. In addition, the existing literature shows that there are very few studies reporting CTX-M-27 and -14 in neonatal samples worldwide. [10],[11]

The enzymatic efficiency of the variants of CTX-M enzymes against different cephalosporins is related to their amino acid sequences. Although both the CTX-M enzymes identified in this study (blaCTX-M-14-like and blaCTX-M-27-like genes) belong to group 9, the deduced amino acid sequence of CTX-M-14 differed from CTX-M-27 by the substitution Asp240Gly. [3] CTX-M-27, which harbours the Asp240Gly substitution, confers eightfold higher levels of resistance to ceftazidime than its parental enzyme, CTX-M-14. [3] This study also shows that the Gly-240-harbouring enzyme, CTX-M-27, confers to E. coli (I-3) a higher MIC of ceftazidime (MIC, 2 μg/ml) than does the Asp-240-harbouring CTX-M-14 enzyme in I-1 (MIC, 0.25 μg/ml).

The gut has been considered to be a reservoir of pathogens and an excellent niche for the emergence of antibiotic resistance. [12] Our study exemplifies this very fact, as the carriage of the ESBL-producing organisms is high and both the isolates with the blaCTX-M-9 group gene were from the neonatal gut. The blaCTX-M-15 was the predominant ESBL gene in this setting. The CTX-M-9 group was isolated from the stool samples and did not cause infection. However, several studies have shown that intestinal colonisation by Gram-negative bacteria precedes the onset of disease. [13] CTX-M genes are plasmid mediated and can easily disseminate by horizontal gene transfer among the different bacterial species. Furthermore, the transmissibility of the CTX-M group 9 genes was also confirmed by conjugation in this study. Thus, the stool isolates, indicative of newer mechanisms of antibiotic resistance, should not be ignored; as such resistant genes could be transmitted.

In conclusion, this study reports the isolation of CTX-M group 9 genes from India and underscores the importance of the survey of the gut flora in evaluating the changing repertoire of organisms and the pattern of antibiotic susceptibilities. Such surveys will also be able to indicate the emergence of new genes. With the constantly increasing number of CTX-M enzymes worldwide and their differential hydrolysis of cephalosporins, the detection of CTX-M group 9 in India is indeed worrisome.

 ~ References Top

1.Qazi SA, Stoll BJ. Neonatal sepsis: A major global public health challenge. Pediatr Infect Dis J 2009;28:S1-2.  Back to cited text no. 1
2.Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: Hospital prevalence and susceptibility patterns. Rev Infect Dis 1988;10:867-77.   Back to cited text no. 2
3.Bonnet R. Growing group of extended-spectrum â-lacamases: The CTX-M enzymes. Antimicrob Agents Chemother 2004;48:1-14.  Back to cited text no. 3
4.Ensor VM, Shahid M, Evans JT, Hawkey PM. Occurrence, prevalence and genetic environment of CTX-M â-lactamases in Enterobacteriaceae from Indian hospitals. J Antimicrob Chemother 2006;58:1260-3.  Back to cited text no. 4
5.Clinical and Laboratory standards institute performance standards for antimicrobial susceptibility tests. Eighteenth informational supplement. Wayne, PA: CLSI document; 2008. M100-S18.  Back to cited text no. 5
6.Munday CJ, Whitehead GM, Todd NJ, Campbell M, Hawkey PM. Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum beta-lactamases in York, UK. J Antimicrob Chemother 2004;54:628-33.   Back to cited text no. 6
7.Goyal A, Prasad KN, Prasad A, Gupta S, Ghoshal U, Ayyagari A. Extended spectrum beta-lactamases in Escherichia coli and Klebsiella pneumoniae and associated risk factors. Indian J Med Res 2009;129:695-700.   Back to cited text no. 7
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8.Kingsley J, Verghese S. Sequence analysis of blaCTX-M-28 , an ESBL responsible for third-generation cephalosporin resistance in Enterobacteriaceae, for the first time in India. Indian J Pathol Microbiol 2008;51:218-21.  Back to cited text no. 8
[PUBMED]  Medknow Journal  
9.Shakil S, Akram M, Ali SM, Khan AU. Acquisition of extended-spectrum {beta}-lactamase producing Escherichia coli strains in male and female infants admitted to a neonatal intensive care unit: Molecular epidemiology and analysis of risk factors. J Med Microbiol 2010;59:948-54.  Back to cited text no. 9
10.Bouallègue-Godet O, Ben Salem Y, Fabre L, Demartin M, Grimont PA, Mzoughi R, et al. Nosocomial Outbreak Caused by Salmonella enterica serotype Livingstone Producing CTX-M-27 Extended-Spectrum â-Lactamase in a Neonatal Unit in Sousse, Tunisia. J Clin Microbiol 2005;43:1037-44.  Back to cited text no. 10
11.Briñas L, Lantero M, Zarazaga M, Pérez F, Torres C. Outbreak of SHV-5 â-Lactamase-Producing Klebsiella pneumoniae in a Neonatal-Pediatric Intensive Care Unit in Spain. Microb Drug Resist 2004;10:354-8.  Back to cited text no. 11
12.Salyers AA, Gupta A, Wang Y. Human intestinal bacteria as reservoirs for antibiotic resistance genes. Trends Microbiol 2004;12:412-6.  Back to cited text no. 12
13.Donskey CJ. Antibiotic regimens and intestinal colonization with antibiotic-resistant gram-negative bacilli. Clin Infect Dis 2006;43:S62-9.  Back to cited text no. 13


  [Figure 1], [Figure 2]

  [Table 1]

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